Phylogenetic inference with q2-phylogeny¶
Phylogenetic inference with q2-phylogeny
Note
This tutorial assumes, you’ve read through the QIIME 2 Overview documentation and have at least worked through some of the other Tutorials.
Inferring phylogenies¶
Several downstream diversity metrics, available within QIIME 2, require that a phylogenetic tree be constructed using the Operational Taxonomic Units (OTUs) or Exact Sequence Variants (ESVs) being investigated.
But how do we proceed to construct a phylogeny from our sequence data?
Well, there are two phylogeny-based approaches we can use. Deciding upon which to use is largely dependent on your study questions:
1. A reference-based fragment insertion approach. Which, is likely the ideal choice. Especially, if your reference phylogeny (and associated representative sequences) encompass neighboring relatives of which your sequences can be reliably inserted. Any sequences that do not match well enough to the reference are not inserted. For example, this approach may not work well if your data contain sequences that are not well represented within your reference phylogeny (e.g. missing clades, etc.). For more information, check out these great fragment insertion examples.
2. A de novo approach. Marker genes that can be globally aligned across divergent taxa, are usually amenable to sequence alignment and phylogenetic investigation through this approach. Be mindful of the length of your sequences when constructing a de novo phylogeny, short reads many not have enough phylogenetic information to capture a meaningful phylogeny. This community tutorial will focus on the de novo approaches.
Here, you will learn how to make use of de novo phylogenetic approaches to:
generate a sequence alignment within QIIME 2
mask the alignment if needed
construct a phylogenetic tree
root the phylogenetic tree
If you would like to substitute any of the steps outlined here by making use of tools external to QIIME 2, please see the import, export, and filtering documentation where appropriate.
Sequence Alignment¶
Prior to constructing a phylogeny we must generate a multiple sequence alignment (MSA). When constructing a MSA we are making a statement about the putative homology of the aligned residues (columns of the MSA) by virtue of their sequence similarity.
The number of algorithms to construct a MSA are legion. We will make use of MAFFT (Multiple Alignment using Fast Fourier Transform)) via the q2-alignment plugin. For more information checkout the MAFFT paper.
Let’s start by creating a directory to work in:
mkdir qiime2-phylogeny-tutorial
cd qiime2-phylogeny-tutorial
Next, download the data:
Download URL: https://data.qiime2.org/2019.10/tutorials/phylogeny/rep-seqs.qza
Save as: rep-seqs.qza
wget \
-O "rep-seqs.qza" \
"https://data.qiime2.org/2019.10/tutorials/phylogeny/rep-seqs.qza"
curl -sL \
"https://data.qiime2.org/2019.10/tutorials/phylogeny/rep-seqs.qza" > \
"rep-seqs.qza"
Run MAFFT
qiime alignment mafft \
--i-sequences rep-seqs.qza \
--o-alignment aligned-rep-seqs.qza
Reducing alignment ambiguity: masking and reference alignments¶
Why mask an alignment?
Masking helps to eliminate alignment columns that are phylogenetically uninformative or misleading before phylogenetic analysis. Much of the time alignment errors can introduce noise and confound phylogenetic inference. It is common practice to mask (remove) these ambiguously aligned regions prior to performing phylogenetic inference. In particular, David Lane’s (1991) chapter 16S/23S rRNA sequencing proposed masking SSU data prior to phylogenetic analysis. However, knowing how to deal with ambiguously aligned regions and when to apply masks largely depends on the marker genes being analyzed and the question being asked of the data.
Note
Keep in mind that this is still an active area of discussion, as highlighted by the following non-exhaustive list of articles: Wu et al. 2012, Ashkenazy et al. 2018, Schloss 2010, Tan et al. 2015, Rajan 2015.
How to mask alignment.
For our purposes, we’ll assume that we have ambiguously aligned columns in the
MAFFT alignment we produced above. The default settings for the
--p-min-conservation
of the
alignment mask approximates the
Lane mask filtering of QIIME 1. Keep an eye out for updates to the alignment
plugin.
qiime alignment mask \
--i-alignment aligned-rep-seqs.qza \
--o-masked-alignment masked-aligned-rep-seqs.qza
Reference based alignments
There are a variety of tools such as PyNAST) (using NAST), Infernal, and SINA, etc., that attempt to reduce the amount of ambiguously aligned regions by using curated reference alignments (e.g. SILVA. Reference alignments are particularly powerful for rRNA gene sequence data, as knowledge of secondary structure is incorporated into the curation process, thus increasing alignment quality. For a more in-depth and eloquent overview of reference-based alignment approaches, check out the great SINA community tutorial).
Note
Alignments constructed using reference based alignment approaches can be masked too, just like the above MAFFT example. Also, the reference alignment approach we are discussing here is distinct from the reference phylogeny approach (i.e. q2-fragment-insertion) we mentioned earlier. That is, we are not inserting our data into an existing tree, but simply trying to create a more robust alignment for making a better de novo phylogeny.
Construct a phylogeny¶
As with MSA algorithms, phylogenetic inference tools are also legion. Fortunately, there are many great resources to learn about phylogentics. Below are just a few introductory resources to get you started:
There are several methods / pipelines available through the q2-phylogeny plugin of :qiime2:. These are based on the following tools:
Methods¶
fasttree¶
FastTree is able to construct phylogenies from large sequence alignments quite rapidly. It does this by using the using a CAT-like rate category approximation, which is also available through RAxML (discussed below). Check out the FastTree online manual for more information.
qiime phylogeny fasttree \
--i-alignment masked-aligned-rep-seqs.qza \
--o-tree fasttree-tree.qza
Tip
For an easy and direct way to view your tree.qza
files, upload
them to iTOL. Here, you can interactively view and manipulate your
phylogeny. Even better, while viewing the tree topology in “Normal mode”,
you can drag and drop your associated alignment.qza
(the one you used to
build the phylogeny) or a relevent taxonomy.qza
file onto the iTOL tree
visualization. This will allow you to directly view the sequence alignment
or taxonomy alongside the phylogeny. 🕶
raxml¶
Like fasttree
, raxml
will perform a single phylogentic inference and
return a tree. Note, the default model for raxml
is
--p-substitution-model GTRGAMMA
. If you’d like to construct a tree using
the CAT model like fasttree
, simply replace GTRGAMMA
with GTRCAT
as
shown below:
qiime phylogeny raxml \
--i-alignment masked-aligned-rep-seqs.qza \
--p-substitution-model GTRCAT \
--o-tree raxml-cat-tree.qza \
--verbose
stdout:
Warning, you specified a working directory via "-w"
Keep in mind that RAxML only accepts absolute path names, not relative ones!
RAxML can't, parse the alignment file as phylip file
it will now try to parse it as FASTA file
Using BFGS method to optimize GTR rate parameters, to disable this specify "--no-bfgs"
This is RAxML version 8.2.12 released by Alexandros Stamatakis on May 2018.
With greatly appreciated code contributions by:
Andre Aberer (HITS)
Simon Berger (HITS)
Alexey Kozlov (HITS)
Kassian Kobert (HITS)
David Dao (KIT and HITS)
Sarah Lutteropp (KIT and HITS)
Nick Pattengale (Sandia)
Wayne Pfeiffer (SDSC)
Akifumi S. Tanabe (NRIFS)
Charlie Taylor (UF)
Alignment has 157 distinct alignment patterns
Proportion of gaps and completely undetermined characters in this alignment: 39.77%
RAxML rapid hill-climbing mode
Using 1 distinct models/data partitions with joint branch length optimization
Executing 1 inferences on the original alignment using 1 distinct randomized MP trees
All free model parameters will be estimated by RAxML
ML estimate of 25 per site rate categories
Likelihood of final tree will be evaluated and optimized under GAMMA
GAMMA Model parameters will be estimated up to an accuracy of 0.1000000000 Log Likelihood units
Partition: 0
Alignment Patterns: 157
Name: No Name Provided
DataType: DNA
Substitution Matrix: GTR
RAxML was called as follows:
raxmlHPC -m GTRCAT -p 6817 -N 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-30edg2qt/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpmcuswmfx -n q2
Partition: 0 with name: No Name Provided
Base frequencies: 0.243 0.182 0.319 0.256
Inference[0]: Time 0.485824 CAT-based likelihood -1258.134859, best rearrangement setting 5
Conducting final model optimizations on all 1 trees under GAMMA-based models ....
Inference[0] final GAMMA-based Likelihood: -1387.511656 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpmcuswmfx/RAxML_result.q2
Starting final GAMMA-based thorough Optimization on tree 0 likelihood -1387.511656 ....
Final GAMMA-based Score of best tree -1387.321440
Program execution info written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpmcuswmfx/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpmcuswmfx/RAxML_bestTree.q2
Overall execution time: 1.051118 secs or 0.000292 hours or 0.000012 days
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.
Command: raxmlHPC -m GTRCAT -p 6817 -N 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-30edg2qt/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpmcuswmfx -n q2
Saved Phylogeny[Unrooted] to: raxml-cat-tree.qza
Perform multiple searches using raxml¶
If you’d like to perform a more thorough search of “tree space” you can
instruct raxml
to perform multiple independent searches on the full
alignment by using --p-n-searches 5
. Once these 5 independent searches are
completed, only the single best scoring tree will be returned. Note, we are
not bootstrapping here, we’ll do that in a later example. Let’s set
--p-substitution-model GTRCAT
. Finally, let’s also manually set a seed via
--p-seed
. By setting our seed, we allow other users the ability to
reproduce our phylogeny. That is, anyone using the same sequence alignment and
substitution model, will generate the same tree as long as they set the same
seed value. Although, --p-seed
is not a required argument, it is generally
a good idea to set this value.
qiime phylogeny raxml \
--i-alignment masked-aligned-rep-seqs.qza \
--p-substitution-model GTRCAT \
--p-seed 1723 \
--p-n-searches 5 \
--o-tree raxml-cat-searches-tree.qza \
--verbose
stdout:
Warning, you specified a working directory via "-w"
Keep in mind that RAxML only accepts absolute path names, not relative ones!
RAxML can't, parse the alignment file as phylip file
it will now try to parse it as FASTA file
Using BFGS method to optimize GTR rate parameters, to disable this specify "--no-bfgs"
This is RAxML version 8.2.12 released by Alexandros Stamatakis on May 2018.
With greatly appreciated code contributions by:
Andre Aberer (HITS)
Simon Berger (HITS)
Alexey Kozlov (HITS)
Kassian Kobert (HITS)
David Dao (KIT and HITS)
Sarah Lutteropp (KIT and HITS)
Nick Pattengale (Sandia)
Wayne Pfeiffer (SDSC)
Akifumi S. Tanabe (NRIFS)
Charlie Taylor (UF)
Alignment has 157 distinct alignment patterns
Proportion of gaps and completely undetermined characters in this alignment: 39.77%
RAxML rapid hill-climbing mode
Using 1 distinct models/data partitions with joint branch length optimization
Executing 5 inferences on the original alignment using 5 distinct randomized MP trees
All free model parameters will be estimated by RAxML
ML estimate of 25 per site rate categories
Likelihood of final tree will be evaluated and optimized under GAMMA
GAMMA Model parameters will be estimated up to an accuracy of 0.1000000000 Log Likelihood units
Partition: 0
Alignment Patterns: 157
Name: No Name Provided
DataType: DNA
Substitution Matrix: GTR
RAxML was called as follows:
raxmlHPC -m GTRCAT -p 1723 -N 5 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-nopa3msr/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwrbcnmci -n q2
Partition: 0 with name: No Name Provided
Base frequencies: 0.243 0.182 0.319 0.256
Inference[0]: Time 0.616283 CAT-based likelihood -1238.242991, best rearrangement setting 5
Inference[1]: Time 0.506815 CAT-based likelihood -1249.502284, best rearrangement setting 5
Inference[2]: Time 0.547314 CAT-based likelihood -1242.978035, best rearrangement setting 5
Inference[3]: Time 0.657815 CAT-based likelihood -1243.159855, best rearrangement setting 5
Inference[4]: Time 0.500320 CAT-based likelihood -1261.321621, best rearrangement setting 5
Conducting final model optimizations on all 5 trees under GAMMA-based models ....
Inference[0] final GAMMA-based Likelihood: -1388.324037 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwrbcnmci/RAxML_result.q2.RUN.0
Inference[1] final GAMMA-based Likelihood: -1392.813982 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwrbcnmci/RAxML_result.q2.RUN.1
Inference[2] final GAMMA-based Likelihood: -1388.073642 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwrbcnmci/RAxML_result.q2.RUN.2
Inference[3] final GAMMA-based Likelihood: -1387.945266 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwrbcnmci/RAxML_result.q2.RUN.3
Inference[4] final GAMMA-based Likelihood: -1387.557031 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwrbcnmci/RAxML_result.q2.RUN.4
Starting final GAMMA-based thorough Optimization on tree 4 likelihood -1387.557031 ....
Final GAMMA-based Score of best tree -1387.385075
Program execution info written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwrbcnmci/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwrbcnmci/RAxML_bestTree.q2
Overall execution time: 3.586809 secs or 0.000996 hours or 0.000042 days
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.
Command: raxmlHPC -m GTRCAT -p 1723 -N 5 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-nopa3msr/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwrbcnmci -n q2
Saved Phylogeny[Unrooted] to: raxml-cat-searches-tree.qza
raxml-rapid-bootstrap¶
In phylogenetics, it is good practice to check how well the splits /
bipartitions in your phylogeny are supported. Often one is interested in
which clades are robustly separated from other clades in the phylogeny. One
way, of doing this is via bootstrapping (See the Bootstrapping section of the
first introductory link above). In QIIME 2, we’ve provided access to the RAxML
rapid bootstrap feature. The only difference between this command and the
previous are the additional flags --p-bootstrap-replicates
and
--p-rapid-bootstrap-seed
. It is quite common to perform anywhere from 100 -
1000 bootstrap replicates. The --p-rapid-bootstrap-seed
works very much
like the --p-seed
argument from above except that it allows anyone to
reproduce the bootstrapping process and the associated supports for your
splits.
As per the RAxML online documentation and the RAxML manual, the rapid bootstrapping command that we will execute below will do the following:
Bootstrap the input alignment 100 times and perform a Maximum Likelihood (ML) search on each.
Find best scoring ML tree through multiple independent searches using the original input alignment. The number of independent searches is determined by the number of bootstrap replicates set in the 1st step. That is, your search becomes more thorough with increasing bootstrap replicates. The ML optimization of RAxML uses every 5th bootstrap tree as the starting tree for an ML search on the original alignment.
Map the bipartitions (bootstrap supports, 1st step) onto the best scoring ML tree (2nd step).
qiime phylogeny raxml-rapid-bootstrap \
--i-alignment masked-aligned-rep-seqs.qza \
--p-seed 1723 \
--p-rapid-bootstrap-seed 9384 \
--p-bootstrap-replicates 100 \
--p-substitution-model GTRCAT \
--o-tree raxml-cat-bootstrap-tree.qza \
--verbose
stdout:
Warning, you specified a working directory via "-w"
Keep in mind that RAxML only accepts absolute path names, not relative ones!
RAxML can't, parse the alignment file as phylip file
it will now try to parse it as FASTA file
Using BFGS method to optimize GTR rate parameters, to disable this specify "--no-bfgs"
This is RAxML version 8.2.12 released by Alexandros Stamatakis on May 2018.
With greatly appreciated code contributions by:
Andre Aberer (HITS)
Simon Berger (HITS)
Alexey Kozlov (HITS)
Kassian Kobert (HITS)
David Dao (KIT and HITS)
Sarah Lutteropp (KIT and HITS)
Nick Pattengale (Sandia)
Wayne Pfeiffer (SDSC)
Akifumi S. Tanabe (NRIFS)
Charlie Taylor (UF)
Alignment has 157 distinct alignment patterns
Proportion of gaps and completely undetermined characters in this alignment: 39.77%
RAxML rapid bootstrapping and subsequent ML search
Using 1 distinct models/data partitions with joint branch length optimization
Executing 100 rapid bootstrap inferences and thereafter a thorough ML search
All free model parameters will be estimated by RAxML
ML estimate of 25 per site rate categories
Likelihood of final tree will be evaluated and optimized under GAMMA
GAMMA Model parameters will be estimated up to an accuracy of 0.1000000000 Log Likelihood units
Partition: 0
Alignment Patterns: 157
Name: No Name Provided
DataType: DNA
Substitution Matrix: GTR
RAxML was called as follows:
raxmlHPC -f a -m GTRCAT -p 1723 -x 9384 -N 100 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-rqm281x4/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpp9_g7icr -n q2bootstrap
Time for BS model parameter optimization 0.051422
Bootstrap[0]: Time 0.180020 seconds, bootstrap likelihood -1199.758796, best rearrangement setting 12
Bootstrap[1]: Time 0.123928 seconds, bootstrap likelihood -1344.229251, best rearrangement setting 6
Bootstrap[2]: Time 0.108201 seconds, bootstrap likelihood -1295.343000, best rearrangement setting 8
Bootstrap[3]: Time 0.094177 seconds, bootstrap likelihood -1273.768320, best rearrangement setting 8
Bootstrap[4]: Time 0.105058 seconds, bootstrap likelihood -1253.402952, best rearrangement setting 6
Bootstrap[5]: Time 0.118345 seconds, bootstrap likelihood -1260.866113, best rearrangement setting 10
Bootstrap[6]: Time 0.112941 seconds, bootstrap likelihood -1293.636299, best rearrangement setting 14
Bootstrap[7]: Time 0.101680 seconds, bootstrap likelihood -1227.178693, best rearrangement setting 6
Bootstrap[8]: Time 0.110852 seconds, bootstrap likelihood -1321.820787, best rearrangement setting 13
Bootstrap[9]: Time 0.117767 seconds, bootstrap likelihood -1147.233446, best rearrangement setting 6
Bootstrap[10]: Time 0.085118 seconds, bootstrap likelihood -1220.766493, best rearrangement setting 13
Bootstrap[11]: Time 0.124581 seconds, bootstrap likelihood -1200.006355, best rearrangement setting 8
Bootstrap[12]: Time 0.132835 seconds, bootstrap likelihood -1346.392834, best rearrangement setting 14
Bootstrap[13]: Time 0.107955 seconds, bootstrap likelihood -1301.111096, best rearrangement setting 14
Bootstrap[14]: Time 0.114613 seconds, bootstrap likelihood -1262.253559, best rearrangement setting 11
Bootstrap[15]: Time 0.114745 seconds, bootstrap likelihood -1215.017551, best rearrangement setting 14
Bootstrap[16]: Time 0.108195 seconds, bootstrap likelihood -1238.832009, best rearrangement setting 7
Bootstrap[17]: Time 0.100415 seconds, bootstrap likelihood -1393.989732, best rearrangement setting 12
Bootstrap[18]: Time 0.104471 seconds, bootstrap likelihood -1173.921002, best rearrangement setting 15
Bootstrap[19]: Time 0.108229 seconds, bootstrap likelihood -1185.726976, best rearrangement setting 11
Bootstrap[20]: Time 0.096617 seconds, bootstrap likelihood -1158.491940, best rearrangement setting 6
Bootstrap[21]: Time 0.093788 seconds, bootstrap likelihood -1154.664272, best rearrangement setting 11
Bootstrap[22]: Time 0.104610 seconds, bootstrap likelihood -1244.159837, best rearrangement setting 10
Bootstrap[23]: Time 0.124368 seconds, bootstrap likelihood -1211.171036, best rearrangement setting 15
Bootstrap[24]: Time 0.105563 seconds, bootstrap likelihood -1261.440677, best rearrangement setting 12
Bootstrap[25]: Time 0.106259 seconds, bootstrap likelihood -1331.836715, best rearrangement setting 15
Bootstrap[26]: Time 0.109827 seconds, bootstrap likelihood -1129.144509, best rearrangement setting 5
Bootstrap[27]: Time 0.137045 seconds, bootstrap likelihood -1226.624056, best rearrangement setting 7
Bootstrap[28]: Time 0.135334 seconds, bootstrap likelihood -1221.046176, best rearrangement setting 12
Bootstrap[29]: Time 0.088339 seconds, bootstrap likelihood -1211.791204, best rearrangement setting 14
Bootstrap[30]: Time 0.106728 seconds, bootstrap likelihood -1389.442380, best rearrangement setting 5
Bootstrap[31]: Time 0.112468 seconds, bootstrap likelihood -1303.638592, best rearrangement setting 12
Bootstrap[32]: Time 0.119762 seconds, bootstrap likelihood -1172.859456, best rearrangement setting 12
Bootstrap[33]: Time 0.105989 seconds, bootstrap likelihood -1244.617135, best rearrangement setting 9
Bootstrap[34]: Time 0.101197 seconds, bootstrap likelihood -1211.871717, best rearrangement setting 15
Bootstrap[35]: Time 0.121801 seconds, bootstrap likelihood -1299.862912, best rearrangement setting 5
Bootstrap[36]: Time 0.097434 seconds, bootstrap likelihood -1141.967505, best rearrangement setting 5
Bootstrap[37]: Time 0.121257 seconds, bootstrap likelihood -1283.923198, best rearrangement setting 12
Bootstrap[38]: Time 0.097948 seconds, bootstrap likelihood -1304.250946, best rearrangement setting 5
Bootstrap[39]: Time 0.089179 seconds, bootstrap likelihood -1407.084376, best rearrangement setting 15
Bootstrap[40]: Time 0.110386 seconds, bootstrap likelihood -1277.946299, best rearrangement setting 13
Bootstrap[41]: Time 0.105007 seconds, bootstrap likelihood -1279.006200, best rearrangement setting 7
Bootstrap[42]: Time 0.104400 seconds, bootstrap likelihood -1160.274606, best rearrangement setting 6
Bootstrap[43]: Time 0.126724 seconds, bootstrap likelihood -1216.079259, best rearrangement setting 14
Bootstrap[44]: Time 0.098282 seconds, bootstrap likelihood -1382.278311, best rearrangement setting 8
Bootstrap[45]: Time 0.110563 seconds, bootstrap likelihood -1099.004439, best rearrangement setting 11
Bootstrap[46]: Time 0.089856 seconds, bootstrap likelihood -1296.527478, best rearrangement setting 8
Bootstrap[47]: Time 0.138094 seconds, bootstrap likelihood -1291.322658, best rearrangement setting 9
Bootstrap[48]: Time 0.087200 seconds, bootstrap likelihood -1161.908080, best rearrangement setting 6
Bootstrap[49]: Time 0.117201 seconds, bootstrap likelihood -1257.348428, best rearrangement setting 13
Bootstrap[50]: Time 0.135139 seconds, bootstrap likelihood -1309.422533, best rearrangement setting 13
Bootstrap[51]: Time 0.098555 seconds, bootstrap likelihood -1197.633097, best rearrangement setting 11
Bootstrap[52]: Time 0.111778 seconds, bootstrap likelihood -1347.123005, best rearrangement setting 8
Bootstrap[53]: Time 0.099514 seconds, bootstrap likelihood -1234.934890, best rearrangement setting 14
Bootstrap[54]: Time 0.118527 seconds, bootstrap likelihood -1227.092434, best rearrangement setting 6
Bootstrap[55]: Time 0.123914 seconds, bootstrap likelihood -1280.635747, best rearrangement setting 7
Bootstrap[56]: Time 0.099109 seconds, bootstrap likelihood -1225.911449, best rearrangement setting 6
Bootstrap[57]: Time 0.089101 seconds, bootstrap likelihood -1236.213347, best rearrangement setting 11
Bootstrap[58]: Time 0.134924 seconds, bootstrap likelihood -1393.245723, best rearrangement setting 14
Bootstrap[59]: Time 0.106722 seconds, bootstrap likelihood -1212.039371, best rearrangement setting 6
Bootstrap[60]: Time 0.093722 seconds, bootstrap likelihood -1248.692011, best rearrangement setting 10
Bootstrap[61]: Time 0.109004 seconds, bootstrap likelihood -1172.820979, best rearrangement setting 13
Bootstrap[62]: Time 0.129389 seconds, bootstrap likelihood -1126.745788, best rearrangement setting 14
Bootstrap[63]: Time 0.099084 seconds, bootstrap likelihood -1267.434444, best rearrangement setting 12
Bootstrap[64]: Time 0.095805 seconds, bootstrap likelihood -1340.680748, best rearrangement setting 5
Bootstrap[65]: Time 0.097272 seconds, bootstrap likelihood -1072.671059, best rearrangement setting 5
Bootstrap[66]: Time 0.123355 seconds, bootstrap likelihood -1234.294838, best rearrangement setting 8
Bootstrap[67]: Time 0.119166 seconds, bootstrap likelihood -1109.249439, best rearrangement setting 15
Bootstrap[68]: Time 0.093235 seconds, bootstrap likelihood -1314.493588, best rearrangement setting 8
Bootstrap[69]: Time 0.095402 seconds, bootstrap likelihood -1173.850035, best rearrangement setting 13
Bootstrap[70]: Time 0.107129 seconds, bootstrap likelihood -1231.066465, best rearrangement setting 10
Bootstrap[71]: Time 0.101057 seconds, bootstrap likelihood -1146.861379, best rearrangement setting 9
Bootstrap[72]: Time 0.087689 seconds, bootstrap likelihood -1148.753369, best rearrangement setting 8
Bootstrap[73]: Time 0.100889 seconds, bootstrap likelihood -1333.374056, best rearrangement setting 9
Bootstrap[74]: Time 0.090034 seconds, bootstrap likelihood -1259.382378, best rearrangement setting 5
Bootstrap[75]: Time 0.097727 seconds, bootstrap likelihood -1319.944496, best rearrangement setting 6
Bootstrap[76]: Time 0.110285 seconds, bootstrap likelihood -1309.042165, best rearrangement setting 14
Bootstrap[77]: Time 0.134228 seconds, bootstrap likelihood -1232.061289, best rearrangement setting 8
Bootstrap[78]: Time 0.114384 seconds, bootstrap likelihood -1261.333984, best rearrangement setting 9
Bootstrap[79]: Time 0.113600 seconds, bootstrap likelihood -1194.644341, best rearrangement setting 13
Bootstrap[80]: Time 0.102618 seconds, bootstrap likelihood -1214.037389, best rearrangement setting 9
Bootstrap[81]: Time 0.110836 seconds, bootstrap likelihood -1224.527657, best rearrangement setting 8
Bootstrap[82]: Time 0.127031 seconds, bootstrap likelihood -1241.464826, best rearrangement setting 11
Bootstrap[83]: Time 0.097515 seconds, bootstrap likelihood -1230.730558, best rearrangement setting 6
Bootstrap[84]: Time 0.100665 seconds, bootstrap likelihood -1219.034592, best rearrangement setting 10
Bootstrap[85]: Time 0.111387 seconds, bootstrap likelihood -1280.071994, best rearrangement setting 8
Bootstrap[86]: Time 0.092283 seconds, bootstrap likelihood -1444.747777, best rearrangement setting 9
Bootstrap[87]: Time 0.094826 seconds, bootstrap likelihood -1245.890035, best rearrangement setting 14
Bootstrap[88]: Time 0.110137 seconds, bootstrap likelihood -1287.832766, best rearrangement setting 7
Bootstrap[89]: Time 0.130631 seconds, bootstrap likelihood -1325.245976, best rearrangement setting 5
Bootstrap[90]: Time 0.116055 seconds, bootstrap likelihood -1227.883697, best rearrangement setting 5
Bootstrap[91]: Time 0.116502 seconds, bootstrap likelihood -1273.489392, best rearrangement setting 8
Bootstrap[92]: Time 0.048078 seconds, bootstrap likelihood -1234.725870, best rearrangement setting 7
Bootstrap[93]: Time 0.119031 seconds, bootstrap likelihood -1235.733064, best rearrangement setting 11
Bootstrap[94]: Time 0.097571 seconds, bootstrap likelihood -1204.319488, best rearrangement setting 15
Bootstrap[95]: Time 0.095067 seconds, bootstrap likelihood -1183.328582, best rearrangement setting 11
Bootstrap[96]: Time 0.111702 seconds, bootstrap likelihood -1196.298898, best rearrangement setting 13
Bootstrap[97]: Time 0.118947 seconds, bootstrap likelihood -1339.251746, best rearrangement setting 12
Bootstrap[98]: Time 0.045710 seconds, bootstrap likelihood -1404.363552, best rearrangement setting 7
Bootstrap[99]: Time 0.058280 seconds, bootstrap likelihood -1270.157811, best rearrangement setting 7
Overall Time for 100 Rapid Bootstraps 10.784172 seconds
Average Time per Rapid Bootstrap 0.107842 seconds
Starting ML Search ...
Fast ML optimization finished
Fast ML search Time: 4.332645 seconds
Slow ML Search 0 Likelihood: -1387.994678
Slow ML Search 1 Likelihood: -1387.994678
Slow ML Search 2 Likelihood: -1387.994676
Slow ML Search 3 Likelihood: -1387.994650
Slow ML Search 4 Likelihood: -1387.994685
Slow ML Search 5 Likelihood: -1388.092954
Slow ML Search 6 Likelihood: -1388.182551
Slow ML Search 7 Likelihood: -1388.182563
Slow ML Search 8 Likelihood: -1388.182547
Slow ML Search 9 Likelihood: -1387.994723
Slow ML optimization finished
Slow ML search Time: 2.209532 seconds
Thorough ML search Time: 0.582538 seconds
Final ML Optimization Likelihood: -1387.204993
Model Information:
Model Parameters of Partition 0, Name: No Name Provided, Type of Data: DNA
alpha: 1.227800
Tree-Length: 7.823400
rate A <-> C: 0.332564
rate A <-> G: 2.312784
rate A <-> T: 2.215466
rate C <-> G: 1.243321
rate C <-> T: 3.278770
rate G <-> T: 1.000000
freq pi(A): 0.243216
freq pi(C): 0.181967
freq pi(G): 0.319196
freq pi(T): 0.255621
ML search took 7.131437 secs or 0.001981 hours
Combined Bootstrap and ML search took 17.915850 secs or 0.004977 hours
Drawing Bootstrap Support Values on best-scoring ML tree ...
Found 1 tree in File /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpp9_g7icr/RAxML_bestTree.q2bootstrap
Found 1 tree in File /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpp9_g7icr/RAxML_bestTree.q2bootstrap
Program execution info written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpp9_g7icr/RAxML_info.q2bootstrap
All 100 bootstrapped trees written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpp9_g7icr/RAxML_bootstrap.q2bootstrap
Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpp9_g7icr/RAxML_bestTree.q2bootstrap
Best-scoring ML tree with support values written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpp9_g7icr/RAxML_bipartitions.q2bootstrap
Best-scoring ML tree with support values as branch labels written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpp9_g7icr/RAxML_bipartitionsBranchLabels.q2bootstrap
Overall execution time for full ML analysis: 17.928981 secs or 0.004980 hours or 0.000208 days
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.
Command: raxmlHPC -f a -m GTRCAT -p 1723 -x 9384 -N 100 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-rqm281x4/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpp9_g7icr -n q2bootstrap
Saved Phylogeny[Unrooted] to: raxml-cat-bootstrap-tree.qza
Tip
Optimizing RAxML Run Time.
You may gave noticed that we haven’t added the flag --p-raxml-version
to
the RAxML methods. This parameter provides a means to access versions of
RAxML that have optimized vector instructions for various modern x86
processor architectures. Paraphrased from the RAxML manual and help
documentation: Firstly, most recent processors will support SSE3 vector
instructions (i.e. will likely support the faster AVX2 vector instructions).
Secondly, these instructions will substantially accelerate the likelihood
and parsimony computations. In general, SSE3 versions will run approximately
40% faster than the standard version. The AVX2 version will run 10-30%
faster than the SSE3 version. Additionally, keep in mind that using more
cores / threads will not necessarily decrease run time. The RAxML manual
suggests using 1 core per ~500 DNA alignment patterns. Alignment pattern
information is usually visible on screen, when the --verbose
option is
used. Additionally, try using a rate category (CAT model; via
--p-substitution-model
), which results in equally good trees as the
GAMMA models and is approximately 4 times faster. See the CAT paper. The
CAT approximation is also Ideal for alignments containing 10,000 or more
taxa, and is very much similar the CAT-like model of FastTree2.
iqtree¶
Similar to the raxml
and raxml-rapid-bootstrap
methods above, we
provide similar functionality for IQ-TREE: iqtree
and
iqtree-ultrafast-bootstrap
. IQ-TREE is unique compared to the fastree
and raxml
options, in that it provides access to 286 models of nucleotide
substitution! IQ-TREE can also determine which of these models best fits your
dataset prior to constructing your tree via its built-in ModelFinder
algorithm. This is the default in QIIME 2, but do not worry, you can set any
one of the 286 models of nucleotide substitution via the
--p-substitution-model
flag, e.g. you can set the model as HKY+I+G
instead of the default MFP
(a basic short-hand for: “build a phylogeny
after determining the best fit model as determined by ModelFinder”). Keep in
mind the additional computational time required for model testing via
ModelFinder.
The simplest way to run the
iqtree command with default
settings and automatic model selection (MFP
) is like so:
qiime phylogeny iqtree \
--i-alignment masked-aligned-rep-seqs.qza \
--o-tree iqt-tree.qza \
--verbose
stdout:
IQ-TREE multicore version 1.6.12 for Mac OS X 64-bit built Aug 15 2019
Developed by Bui Quang Minh, Nguyen Lam Tung, Olga Chernomor,
Heiko Schmidt, Dominik Schrempf, Michael Woodhams.
Host: ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-lc6ec9s2/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwa02_fgr/q2iqtree -nt 1
Seed: 914763 (Using SPRNG - Scalable Parallel Random Number Generator)
Time: Fri Nov 1 07:40:44 2019
Kernel: AVX - 1 threads (8 CPU cores detected)
HINT: Use -nt option to specify number of threads because your CPU has 8 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.
Reading alignment file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-lc6ec9s2/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
Gap/Ambiguity Composition p-value
1 e84fcf85a6a4065231dcf343bb862f1cb32abae6 40.65% passed 90.91%
2 5525fb6dab7b6577960147574465990c6df070ad 42.99% passed 99.80%
3 eb3564a35320b53cef22a77288838c7446357327 42.99% passed 25.49%
4 418f1d469f08c99976b313028cf6d3f18f61dd55 43.93% passed 71.86%
5 2e3b2c075901640c4de739473f9246385430b1ed 31.31% passed 90.76%
6 0469f8d819bd45c7638d1c8b0895270a05f34267 38.79% passed 92.82%
7 d162ed685007f5adede58f14aece31dfa1b60c18 40.65% passed 97.17%
8 1d45b2bce36cd995c5dcb755babf512e612ce8b9 41.59% passed 39.04%
9 5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8 31.31% passed 87.21%
10 206656bec2abdbc4aee37a661ef5f4a62b5dd6ae 42.99% passed 85.00%
11 606c23e79bb730ad74e3c6efd72004c36674c17a 47.20% passed 87.78%
12 682e91d7e510ab134d0625234ad224f647c14eb0 41.59% passed 31.01%
13 6a36152105590b1eb095b9503e8f1f226fc73e43 39.25% passed 86.29%
14 6ca685c39a33bfbcb3123129e7af88d573df7d6f 42.06% failed 0.02%
15 8a1c44eb462ed58b21f3fdd72dd22bb657db2980 31.78% passed 54.40%
16 9b220cae8d375ea38b8b481cb95949cda8722fcb 36.92% passed 88.78%
17 aa4698d2e2b1fa71d08e2934a923aad7374a18f6 37.85% passed 90.52%
18 b31aa3f04bc9d5e2498d45cf1983dfaf09faa258 31.78% passed 72.69%
19 d44b129a6181f052198bda3813f0802a91612441 41.59% passed 41.69%
20 ed1acad8a98e8579a44370733533ad7d3fed8006 48.13% passed 58.15%
**** TOTAL 39.77% 1 sequences failed composition chi2 test (p-value<5%; df=3)
Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test 286 DNA models (sample size: 214) ...

Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1418.951
2. Current log-likelihood: -1397.991
3. Current log-likelihood: -1393.472
4. Current log-likelihood: -1392.886
5. Current log-likelihood: -1392.619
6. Current log-likelihood: -1392.468
Optimal log-likelihood: -1392.377
Rate parameters: A-C: 0.14236 A-G: 1.85682 A-T: 1.51945 C-G: 0.73999 C-T: 1.85682 G-T: 1.00000
Base frequencies: A: 0.250 C: 0.250 G: 0.250 T: 0.250
Site proportion and rates: (0.672,0.382) (0.328,2.264)
Parameters optimization took 6 rounds (0.035 sec)
Computing ML distances based on estimated model parameters... 0.002 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1389.227
--------------------------------------------------------------------
| INITIALIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.091 second
Computing log-likelihood of 98 initial trees ... 0.144 seconds
Current best score: -1389.227
Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1382.389
UPDATE BEST LOG-LIKELIHOOD: -1382.389
Iteration 10 / LogL: -1382.398 / Time: 0h:0m:0s
Iteration 20 / LogL: -1382.391 / Time: 0h:0m:0s
Finish initializing candidate tree set (1)
Current best tree score: -1382.389 / CPU time: 0.485
Number of iterations: 20
--------------------------------------------------------------------
| OPTIMIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Iteration 30 / LogL: -1391.358 / Time: 0h:0m:0s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.388
Iteration 40 / LogL: -1403.595 / Time: 0h:0m:0s (0h:0m:1s left)
Iteration 50 / LogL: -1382.521 / Time: 0h:0m:1s (0h:0m:1s left)
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 53: -1382.119
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 55: -1382.044
Iteration 60 / LogL: -1382.069 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 70 / LogL: -1391.264 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 80 / LogL: -1382.445 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 90 / LogL: -1382.096 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 100 / LogL: -1382.402 / Time: 0h:0m:1s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.043
Iteration 110 / LogL: -1382.518 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 120 / LogL: -1382.607 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 130 / LogL: -1382.445 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 140 / LogL: -1382.222 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 150 / LogL: -1382.443 / Time: 0h:0m:2s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 156 ITERATIONS / Time: 0h:0m:2s
--------------------------------------------------------------------
| FINALIZING TREE SEARCH |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1382.043
Optimal log-likelihood: -1382.037
Rate parameters: A-C: 0.18887 A-G: 1.83050 A-T: 1.53116 C-G: 0.77209 C-T: 1.83050 G-T: 1.00000
Base frequencies: A: 0.250 C: 0.250 G: 0.250 T: 0.250
Site proportion and rates: (0.719,0.420) (0.281,2.487)
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1382.037
Total tree length: 6.919
Total number of iterations: 156
CPU time used for tree search: 2.585 sec (0h:0m:2s)
Wall-clock time used for tree search: 2.588 sec (0h:0m:2s)
Total CPU time used: 2.640 sec (0h:0m:2s)
Total wall-clock time used: 2.646 sec (0h:0m:2s)
Analysis results written to:
IQ-TREE report: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwa02_fgr/q2iqtree.iqtree
Maximum-likelihood tree: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwa02_fgr/q2iqtree.treefile
Likelihood distances: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwa02_fgr/q2iqtree.mldist
Screen log file: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwa02_fgr/q2iqtree.log
Date and Time: Fri Nov 1 07:40:48 2019
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-lc6ec9s2/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwa02_fgr/q2iqtree -nt 1
Saved Phylogeny[Unrooted] to: iqt-tree.qza
Specifying a substitution model¶
We can also set a substitution model of our choosing. You may have noticed
while watching the onscreen output of the previous command that the best
fitting model selected by ModelFinder is noted. For the sake of argument, let’s
say the best selected model was shown as GTR+F+I+G4
. The F
is only a
notation to let us know that if a given model supports unequal base
frequencies, then the empirical base frequencies will be used by default.
Using empirical base frequencies (F
), rather than estimating them, greatly
reduces computational time. The iqtree
plugin will not accept F
within
the model notation supplied at the command line, as this will always be implied
automatically for the appropriate model. Also, the iqtree
plugin only
accepts G
not G4
to be specified within the model notation. The 4
is simply another explicit notation to remind us that four rate categories are
being assumed by default. The notation approach used by the plugin simply helps
to retain simplicity and familiarity when supplying model notations on the
command line. So, in brief, we only have to type GTR+I+G
as our input
model:
qiime phylogeny iqtree \
--i-alignment masked-aligned-rep-seqs.qza \
--p-substitution-model 'GTR+I+G' \
--o-tree iqt-gtrig-tree.qza \
--verbose
stdout:
IQ-TREE multicore version 1.6.12 for Mac OS X 64-bit built Aug 15 2019
Developed by Bui Quang Minh, Nguyen Lam Tung, Olga Chernomor,
Heiko Schmidt, Dominik Schrempf, Michael Woodhams.
Host: ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-67hgp5u_/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpbdvurvku/q2iqtree -nt 1
Seed: 791263 (Using SPRNG - Scalable Parallel Random Number Generator)
Time: Fri Nov 1 07:40:52 2019
Kernel: AVX - 1 threads (8 CPU cores detected)
HINT: Use -nt option to specify number of threads because your CPU has 8 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.
Reading alignment file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-67hgp5u_/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
Gap/Ambiguity Composition p-value
1 e84fcf85a6a4065231dcf343bb862f1cb32abae6 40.65% passed 90.91%
2 5525fb6dab7b6577960147574465990c6df070ad 42.99% passed 99.80%
3 eb3564a35320b53cef22a77288838c7446357327 42.99% passed 25.49%
4 418f1d469f08c99976b313028cf6d3f18f61dd55 43.93% passed 71.86%
5 2e3b2c075901640c4de739473f9246385430b1ed 31.31% passed 90.76%
6 0469f8d819bd45c7638d1c8b0895270a05f34267 38.79% passed 92.82%
7 d162ed685007f5adede58f14aece31dfa1b60c18 40.65% passed 97.17%
8 1d45b2bce36cd995c5dcb755babf512e612ce8b9 41.59% passed 39.04%
9 5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8 31.31% passed 87.21%
10 206656bec2abdbc4aee37a661ef5f4a62b5dd6ae 42.99% passed 85.00%
11 606c23e79bb730ad74e3c6efd72004c36674c17a 47.20% passed 87.78%
12 682e91d7e510ab134d0625234ad224f647c14eb0 41.59% passed 31.01%
13 6a36152105590b1eb095b9503e8f1f226fc73e43 39.25% passed 86.29%
14 6ca685c39a33bfbcb3123129e7af88d573df7d6f 42.06% failed 0.02%
15 8a1c44eb462ed58b21f3fdd72dd22bb657db2980 31.78% passed 54.40%
16 9b220cae8d375ea38b8b481cb95949cda8722fcb 36.92% passed 88.78%
17 aa4698d2e2b1fa71d08e2934a923aad7374a18f6 37.85% passed 90.52%
18 b31aa3f04bc9d5e2498d45cf1983dfaf09faa258 31.78% passed 72.69%
19 d44b129a6181f052198bda3813f0802a91612441 41.59% passed 41.69%
20 ed1acad8a98e8579a44370733533ad7d3fed8006 48.13% passed 58.15%
**** TOTAL 39.77% 1 sequences failed composition chi2 test (p-value<5%; df=3)
Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
Thoroughly optimizing +I+G parameters from 10 start values...
Init pinv, alpha: 0.000, 1.000 / Estimate: 0.000, 1.281 / LogL: -1392.558
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.008, 1.381 / LogL: -1392.827
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.009, 1.392 / LogL: -1392.900
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.009, 1.387 / LogL: -1392.892
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.008, 1.383 / LogL: -1392.858
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.387 / LogL: -1392.886
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.008, 1.378 / LogL: -1392.831
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.382 / LogL: -1392.845
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.008, 1.382 / LogL: -1392.850
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.008, 1.383 / LogL: -1392.860
Optimal pinv,alpha: 0.000, 1.281 / LogL: -1392.558
Parameters optimization took 0.480 sec
Computing ML distances based on estimated model parameters... 0.003 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1392.710
--------------------------------------------------------------------
| INITIALIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.089 second
Computing log-likelihood of 98 initial trees ... 0.199 seconds
Current best score: -1392.558
Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.259
Iteration 10 / LogL: -1387.287 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.269 / Time: 0h:0m:1s
Finish initializing candidate tree set (2)
Current best tree score: -1387.259 / CPU time: 0.599
Number of iterations: 20
--------------------------------------------------------------------
| OPTIMIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Iteration 30 / LogL: -1396.818 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 40 / LogL: -1387.262 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 50 / LogL: -1387.350 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 60 / LogL: -1387.451 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 70 / LogL: -1406.372 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 80 / LogL: -1392.423 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 90 / LogL: -1387.352 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 100 / LogL: -1387.350 / Time: 0h:0m:3s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 102 ITERATIONS / Time: 0h:0m:3s
--------------------------------------------------------------------
| FINALIZING TREE SEARCH |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.259
Optimal log-likelihood: -1387.253
Rate parameters: A-C: 0.33196 A-G: 2.27261 A-T: 2.15113 C-G: 1.18143 C-T: 3.30120 G-T: 1.00000
Base frequencies: A: 0.243 C: 0.182 G: 0.319 T: 0.256
Proportion of invariable sites: 0.000
Gamma shape alpha: 1.324
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1387.253
Total tree length: 6.714
Total number of iterations: 102
CPU time used for tree search: 3.027 sec (0h:0m:3s)
Wall-clock time used for tree search: 3.030 sec (0h:0m:3s)
Total CPU time used: 3.524 sec (0h:0m:3s)
Total wall-clock time used: 3.538 sec (0h:0m:3s)
Analysis results written to:
IQ-TREE report: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpbdvurvku/q2iqtree.iqtree
Maximum-likelihood tree: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpbdvurvku/q2iqtree.treefile
Likelihood distances: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpbdvurvku/q2iqtree.mldist
Screen log file: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpbdvurvku/q2iqtree.log
Date and Time: Fri Nov 1 07:40:56 2019
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-67hgp5u_/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpbdvurvku/q2iqtree -nt 1
Saved Phylogeny[Unrooted] to: iqt-gtrig-tree.qza
Let’s rerun the command above and add the --p-fast
option. This option,
only compatible with the iqtree
method, resembles the fast search performed
by fasttree
. 🏎 Secondly, let’s also perform multiple tree searches and
keep the best of those trees (as we did earlier with the
raxml --p-n-searches ...
command):
qiime phylogeny iqtree \
--i-alignment masked-aligned-rep-seqs.qza \
--p-substitution-model 'GTR+I+G' \
--p-fast \
--p-n-runs 10 \
--o-tree iqt-gtrig-fast-ms-tree.qza \
--verbose
stdout:
IQ-TREE multicore version 1.6.12 for Mac OS X 64-bit built Aug 15 2019
Developed by Bui Quang Minh, Nguyen Lam Tung, Olga Chernomor,
Heiko Schmidt, Dominik Schrempf, Michael Woodhams.
Host: ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 10 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-7bbn7mkx/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1gz3ic6z/q2iqtree -nt 1 -fast
Seed: 589875 (Using SPRNG - Scalable Parallel Random Number Generator)
Time: Fri Nov 1 07:41:00 2019
Kernel: AVX - 1 threads (8 CPU cores detected)
HINT: Use -nt option to specify number of threads because your CPU has 8 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.
Reading alignment file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-7bbn7mkx/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
Gap/Ambiguity Composition p-value
1 e84fcf85a6a4065231dcf343bb862f1cb32abae6 40.65% passed 90.91%
2 5525fb6dab7b6577960147574465990c6df070ad 42.99% passed 99.80%
3 eb3564a35320b53cef22a77288838c7446357327 42.99% passed 25.49%
4 418f1d469f08c99976b313028cf6d3f18f61dd55 43.93% passed 71.86%
5 2e3b2c075901640c4de739473f9246385430b1ed 31.31% passed 90.76%
6 0469f8d819bd45c7638d1c8b0895270a05f34267 38.79% passed 92.82%
7 d162ed685007f5adede58f14aece31dfa1b60c18 40.65% passed 97.17%
8 1d45b2bce36cd995c5dcb755babf512e612ce8b9 41.59% passed 39.04%
9 5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8 31.31% passed 87.21%
10 206656bec2abdbc4aee37a661ef5f4a62b5dd6ae 42.99% passed 85.00%
11 606c23e79bb730ad74e3c6efd72004c36674c17a 47.20% passed 87.78%
12 682e91d7e510ab134d0625234ad224f647c14eb0 41.59% passed 31.01%
13 6a36152105590b1eb095b9503e8f1f226fc73e43 39.25% passed 86.29%
14 6ca685c39a33bfbcb3123129e7af88d573df7d6f 42.06% failed 0.02%
15 8a1c44eb462ed58b21f3fdd72dd22bb657db2980 31.78% passed 54.40%
16 9b220cae8d375ea38b8b481cb95949cda8722fcb 36.92% passed 88.78%
17 aa4698d2e2b1fa71d08e2934a923aad7374a18f6 37.85% passed 90.52%
18 b31aa3f04bc9d5e2498d45cf1983dfaf09faa258 31.78% passed 72.69%
19 d44b129a6181f052198bda3813f0802a91612441 41.59% passed 41.69%
20 ed1acad8a98e8579a44370733533ad7d3fed8006 48.13% passed 58.15%
**** TOTAL 39.77% 1 sequences failed composition chi2 test (p-value<5%; df=3)
---> START RUN NUMBER 1 (seed: 589875)
Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
Thoroughly optimizing +I+G parameters from 10 start values...
Init pinv, alpha: 0.000, 1.000 / Estimate: 0.000, 1.243 / LogL: -1394.457
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.018, 1.300 / LogL: -1395.193
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.023, 1.343 / LogL: -1395.423
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.022, 1.352 / LogL: -1395.409
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.025, 1.352 / LogL: -1395.581
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.027, 1.353 / LogL: -1395.694
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.023, 1.350 / LogL: -1395.451
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.024, 1.352 / LogL: -1395.506
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.025, 1.352 / LogL: -1395.553
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.026, 1.353 / LogL: -1395.597
Optimal pinv,alpha: 0.000, 1.243 / LogL: -1394.457
Parameters optimization took 0.305 sec
Computing ML distances based on estimated model parameters... 0.003 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1392.886
--------------------------------------------------------------------
| INITIALIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.284
Finish initializing candidate tree set (3)
Current best tree score: -1387.284 / CPU time: 0.059
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s
--------------------------------------------------------------------
| FINALIZING TREE SEARCH |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1387.284
Optimal log-likelihood: -1387.262
Rate parameters: A-C: 0.33572 A-G: 2.25727 A-T: 2.13258 C-G: 1.17706 C-T: 3.27735 G-T: 1.00000
Base frequencies: A: 0.243 C: 0.182 G: 0.319 T: 0.256
Proportion of invariable sites: 0.000
Gamma shape alpha: 1.306
Parameters optimization took 1 rounds (0.005 sec)
BEST SCORE FOUND : -1387.262
Total tree length: 6.787
Total number of iterations: 2
CPU time used for tree search: 0.059 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.060 sec (0h:0m:0s)
Total CPU time used: 0.388 sec (0h:0m:0s)
Total wall-clock time used: 0.392 sec (0h:0m:0s)
---> START RUN NUMBER 2 (seed: 590875)
Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.000 seconds
NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1481.570
2. Current log-likelihood: -1401.790
3. Current log-likelihood: -1396.547
4. Current log-likelihood: -1395.118
5. Current log-likelihood: -1394.384
Optimal log-likelihood: -1393.813
Rate parameters: A-C: 0.27488 A-G: 2.41340 A-T: 2.17693 C-G: 1.25270 C-T: 3.29513 G-T: 1.00000
Base frequencies: A: 0.243 C: 0.182 G: 0.319 T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.421
Parameters optimization took 5 rounds (0.037 sec)
Computing ML distances based on estimated model parameters... 0.003 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.791
--------------------------------------------------------------------
| INITIALIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.211
Finish initializing candidate tree set (3)
Current best tree score: -1388.211 / CPU time: 0.046
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s
--------------------------------------------------------------------
| FINALIZING TREE SEARCH |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.211
2. Current log-likelihood: -1388.012
3. Current log-likelihood: -1387.866
4. Current log-likelihood: -1387.758
5. Current log-likelihood: -1387.675
6. Current log-likelihood: -1387.610
7. Current log-likelihood: -1387.559
Optimal log-likelihood: -1387.518
Rate parameters: A-C: 0.35582 A-G: 2.35557 A-T: 2.14271 C-G: 1.20481 C-T: 3.37552 G-T: 1.00000
Base frequencies: A: 0.243 C: 0.182 G: 0.319 T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.361
Parameters optimization took 7 rounds (0.027 sec)
BEST SCORE FOUND : -1387.518
Total tree length: 6.815
Total number of iterations: 2
CPU time used for tree search: 0.045 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.046 sec (0h:0m:0s)
Total CPU time used: 0.129 sec (0h:0m:0s)
Total wall-clock time used: 0.132 sec (0h:0m:0s)
---> START RUN NUMBER 3 (seed: 591875)
Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1480.521
2. Current log-likelihood: -1403.150
3. Current log-likelihood: -1398.384
4. Current log-likelihood: -1396.968
5. Current log-likelihood: -1396.253
Optimal log-likelihood: -1395.745
Rate parameters: A-C: 0.23809 A-G: 2.08501 A-T: 1.96789 C-G: 1.08192 C-T: 2.78800 G-T: 1.00000
Base frequencies: A: 0.243 C: 0.182 G: 0.319 T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.364
Parameters optimization took 5 rounds (0.040 sec)
Computing ML distances based on estimated model parameters... 0.003 sec
WARNING: Some pairwise ML distances are too long (saturated)
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.993
--------------------------------------------------------------------
| INITIALIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.951
Finish initializing candidate tree set (4)
Current best tree score: -1387.951 / CPU time: 0.078
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s
--------------------------------------------------------------------
| FINALIZING TREE SEARCH |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1387.951
2. Current log-likelihood: -1387.792
3. Current log-likelihood: -1387.673
4. Current log-likelihood: -1387.583
5. Current log-likelihood: -1387.515
6. Current log-likelihood: -1387.462
Optimal log-likelihood: -1387.420
Rate parameters: A-C: 0.33363 A-G: 2.24758 A-T: 2.12142 C-G: 1.16644 C-T: 3.24795 G-T: 1.00000
Base frequencies: A: 0.243 C: 0.182 G: 0.319 T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.358
Parameters optimization took 6 rounds (0.024 sec)
BEST SCORE FOUND : -1387.420
Total tree length: 6.700
Total number of iterations: 2
CPU time used for tree search: 0.078 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.078 sec (0h:0m:0s)
Total CPU time used: 0.161 sec (0h:0m:0s)
Total wall-clock time used: 0.164 sec (0h:0m:0s)
---> START RUN NUMBER 4 (seed: 592875)
Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1480.586
2. Current log-likelihood: -1403.151
3. Current log-likelihood: -1398.384
4. Current log-likelihood: -1396.968
5. Current log-likelihood: -1396.253
Optimal log-likelihood: -1395.745
Rate parameters: A-C: 0.23809 A-G: 2.08501 A-T: 1.96788 C-G: 1.08193 C-T: 2.78798 G-T: 1.00000
Base frequencies: A: 0.243 C: 0.182 G: 0.319 T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.364
Parameters optimization took 5 rounds (0.044 sec)
Computing ML distances based on estimated model parameters... 0.003 sec
WARNING: Some pairwise ML distances are too long (saturated)
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.993
--------------------------------------------------------------------
| INITIALIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.951
Finish initializing candidate tree set (4)
Current best tree score: -1387.951 / CPU time: 0.080
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s
--------------------------------------------------------------------
| FINALIZING TREE SEARCH |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1387.951
2. Current log-likelihood: -1387.792
3. Current log-likelihood: -1387.673
4. Current log-likelihood: -1387.583
5. Current log-likelihood: -1387.515
6. Current log-likelihood: -1387.462
Optimal log-likelihood: -1387.420
Rate parameters: A-C: 0.33363 A-G: 2.24758 A-T: 2.12142 C-G: 1.16644 C-T: 3.24795 G-T: 1.00000
Base frequencies: A: 0.243 C: 0.182 G: 0.319 T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.358
Parameters optimization took 6 rounds (0.024 sec)
BEST SCORE FOUND : -1387.420
Total tree length: 6.700
Total number of iterations: 2
CPU time used for tree search: 0.079 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.080 sec (0h:0m:0s)
Total CPU time used: 0.163 sec (0h:0m:0s)
Total wall-clock time used: 0.166 sec (0h:0m:0s)
---> START RUN NUMBER 5 (seed: 593875)
Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1480.521
2. Current log-likelihood: -1403.150
3. Current log-likelihood: -1398.384
4. Current log-likelihood: -1396.968
5. Current log-likelihood: -1396.253
Optimal log-likelihood: -1395.745
Rate parameters: A-C: 0.23809 A-G: 2.08501 A-T: 1.96789 C-G: 1.08192 C-T: 2.78800 G-T: 1.00000
Base frequencies: A: 0.243 C: 0.182 G: 0.319 T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.364
Parameters optimization took 5 rounds (0.036 sec)
Computing ML distances based on estimated model parameters... 0.003 sec
WARNING: Some pairwise ML distances are too long (saturated)
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.993
--------------------------------------------------------------------
| INITIALIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.951
Finish initializing candidate tree set (4)
Current best tree score: -1387.951 / CPU time: 0.082
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s
--------------------------------------------------------------------
| FINALIZING TREE SEARCH |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1387.951
2. Current log-likelihood: -1387.792
3. Current log-likelihood: -1387.673
4. Current log-likelihood: -1387.583
5. Current log-likelihood: -1387.515
6. Current log-likelihood: -1387.462
Optimal log-likelihood: -1387.420
Rate parameters: A-C: 0.33363 A-G: 2.24758 A-T: 2.12142 C-G: 1.16644 C-T: 3.24795 G-T: 1.00000
Base frequencies: A: 0.243 C: 0.182 G: 0.319 T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.358
Parameters optimization took 6 rounds (0.021 sec)
BEST SCORE FOUND : -1387.420
Total tree length: 6.700
Total number of iterations: 2
CPU time used for tree search: 0.081 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.082 sec (0h:0m:0s)
Total CPU time used: 0.158 sec (0h:0m:0s)
Total wall-clock time used: 0.161 sec (0h:0m:0s)
---> START RUN NUMBER 6 (seed: 594875)
Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1481.723
2. Current log-likelihood: -1403.056
3. Current log-likelihood: -1398.274
4. Current log-likelihood: -1396.896
5. Current log-likelihood: -1396.165
Optimal log-likelihood: -1395.664
Rate parameters: A-C: 0.23525 A-G: 2.07316 A-T: 1.96748 C-G: 1.07231 C-T: 2.84580 G-T: 1.00000
Base frequencies: A: 0.243 C: 0.182 G: 0.319 T: 0.256
Proportion of invariable sites: 0.026
Gamma shape alpha: 1.337
Parameters optimization took 5 rounds (0.034 sec)
Computing ML distances based on estimated model parameters... 0.003 sec
WARNING: Some pairwise ML distances are too long (saturated)
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.979
--------------------------------------------------------------------
| INITIALIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.172
Finish initializing candidate tree set (3)
Current best tree score: -1388.172 / CPU time: 0.038
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s
--------------------------------------------------------------------
| FINALIZING TREE SEARCH |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.172
2. Current log-likelihood: -1387.951
3. Current log-likelihood: -1387.788
4. Current log-likelihood: -1387.670
5. Current log-likelihood: -1387.581
6. Current log-likelihood: -1387.513
7. Current log-likelihood: -1387.460
Optimal log-likelihood: -1387.418
Rate parameters: A-C: 0.33576 A-G: 2.25513 A-T: 2.12939 C-G: 1.17073 C-T: 3.26109 G-T: 1.00000
Base frequencies: A: 0.243 C: 0.182 G: 0.319 T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.356
Parameters optimization took 7 rounds (0.028 sec)
BEST SCORE FOUND : -1387.418
Total tree length: 6.706
Total number of iterations: 2
CPU time used for tree search: 0.038 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.039 sec (0h:0m:0s)
Total CPU time used: 0.122 sec (0h:0m:0s)
Total wall-clock time used: 0.125 sec (0h:0m:0s)
---> START RUN NUMBER 7 (seed: 595875)
Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1480.860
2. Current log-likelihood: -1401.866
3. Current log-likelihood: -1396.558
4. Current log-likelihood: -1395.110
5. Current log-likelihood: -1394.382
Optimal log-likelihood: -1393.810
Rate parameters: A-C: 0.27492 A-G: 2.41474 A-T: 2.17844 C-G: 1.25256 C-T: 3.29094 G-T: 1.00000
Base frequencies: A: 0.243 C: 0.182 G: 0.319 T: 0.256
Proportion of invariable sites: 0.026
Gamma shape alpha: 1.421
Parameters optimization took 5 rounds (0.036 sec)
Computing ML distances based on estimated model parameters... 0.003 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.790
--------------------------------------------------------------------
| INITIALIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.211
Finish initializing candidate tree set (3)
Current best tree score: -1388.211 / CPU time: 0.048
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s
--------------------------------------------------------------------
| FINALIZING TREE SEARCH |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.211
2. Current log-likelihood: -1388.012
3. Current log-likelihood: -1387.866
4. Current log-likelihood: -1387.758
5. Current log-likelihood: -1387.675
6. Current log-likelihood: -1387.610
7. Current log-likelihood: -1387.559
Optimal log-likelihood: -1387.518
Rate parameters: A-C: 0.35576 A-G: 2.35519 A-T: 2.14234 C-G: 1.20461 C-T: 3.37491 G-T: 1.00000
Base frequencies: A: 0.243 C: 0.182 G: 0.319 T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.361
Parameters optimization took 7 rounds (0.027 sec)
BEST SCORE FOUND : -1387.518
Total tree length: 6.815
Total number of iterations: 2
CPU time used for tree search: 0.048 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.049 sec (0h:0m:0s)
Total CPU time used: 0.127 sec (0h:0m:0s)
Total wall-clock time used: 0.130 sec (0h:0m:0s)
---> START RUN NUMBER 8 (seed: 596875)
Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.000 seconds
NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1482.403
2. Current log-likelihood: -1404.572
3. Current log-likelihood: -1399.236
4. Current log-likelihood: -1397.831
5. Current log-likelihood: -1397.061
Optimal log-likelihood: -1396.470
Rate parameters: A-C: 0.24622 A-G: 2.08130 A-T: 1.99445 C-G: 1.06444 C-T: 2.85652 G-T: 1.00000
Base frequencies: A: 0.243 C: 0.182 G: 0.319 T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.437
Parameters optimization took 5 rounds (0.041 sec)
Computing ML distances based on estimated model parameters... 0.003 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.961
--------------------------------------------------------------------
| INITIALIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.181
Finish initializing candidate tree set (3)
Current best tree score: -1388.181 / CPU time: 0.055
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s
--------------------------------------------------------------------
| FINALIZING TREE SEARCH |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.181
2. Current log-likelihood: -1387.955
3. Current log-likelihood: -1387.793
4. Current log-likelihood: -1387.674
5. Current log-likelihood: -1387.584
6. Current log-likelihood: -1387.516
7. Current log-likelihood: -1387.463
Optimal log-likelihood: -1387.421
Rate parameters: A-C: 0.33228 A-G: 2.23486 A-T: 2.10851 C-G: 1.15950 C-T: 3.22710 G-T: 1.00000
Base frequencies: A: 0.243 C: 0.182 G: 0.319 T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.360
Parameters optimization took 7 rounds (0.028 sec)
BEST SCORE FOUND : -1387.421
Total tree length: 6.696
Total number of iterations: 2
CPU time used for tree search: 0.055 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.055 sec (0h:0m:0s)
Total CPU time used: 0.143 sec (0h:0m:0s)
Total wall-clock time used: 0.146 sec (0h:0m:0s)
---> START RUN NUMBER 9 (seed: 597875)
Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1482.977
2. Current log-likelihood: -1402.079
3. Current log-likelihood: -1396.806
4. Current log-likelihood: -1395.378
5. Current log-likelihood: -1394.645
Optimal log-likelihood: -1394.074
Rate parameters: A-C: 0.27526 A-G: 2.39376 A-T: 2.12146 C-G: 1.20995 C-T: 3.30275 G-T: 1.00000
Base frequencies: A: 0.243 C: 0.182 G: 0.319 T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.394
Parameters optimization took 5 rounds (0.039 sec)
Computing ML distances based on estimated model parameters... 0.003 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.804
--------------------------------------------------------------------
| INITIALIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.216
BETTER TREE FOUND at iteration 2: -1388.181
Finish initializing candidate tree set (4)
Current best tree score: -1388.181 / CPU time: 0.063
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s
--------------------------------------------------------------------
| FINALIZING TREE SEARCH |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.181
2. Current log-likelihood: -1387.974
3. Current log-likelihood: -1387.831
4. Current log-likelihood: -1387.726
5. Current log-likelihood: -1387.646
6. Current log-likelihood: -1387.585
Optimal log-likelihood: -1387.535
Rate parameters: A-C: 0.36870 A-G: 2.32160 A-T: 2.12854 C-G: 1.22861 C-T: 3.29569 G-T: 1.00000
Base frequencies: A: 0.243 C: 0.182 G: 0.319 T: 0.256
Proportion of invariable sites: 0.006
Gamma shape alpha: 1.331
Parameters optimization took 6 rounds (0.022 sec)
BEST SCORE FOUND : -1387.535
Total tree length: 7.507
Total number of iterations: 2
CPU time used for tree search: 0.062 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.063 sec (0h:0m:0s)
Total CPU time used: 0.143 sec (0h:0m:0s)
Total wall-clock time used: 0.146 sec (0h:0m:0s)
---> START RUN NUMBER 10 (seed: 598875)
Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.000 seconds
NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1480.110
2. Current log-likelihood: -1403.534
3. Current log-likelihood: -1398.492
4. Current log-likelihood: -1397.024
5. Current log-likelihood: -1396.211
Optimal log-likelihood: -1395.651
Rate parameters: A-C: 0.23242 A-G: 2.05799 A-T: 1.95257 C-G: 1.07461 C-T: 2.81853 G-T: 1.00000
Base frequencies: A: 0.243 C: 0.182 G: 0.319 T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.346
Parameters optimization took 5 rounds (0.040 sec)
Computing ML distances based on estimated model parameters... 0.003 sec
WARNING: Some pairwise ML distances are too long (saturated)
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.989
--------------------------------------------------------------------
| INITIALIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.946
Finish initializing candidate tree set (4)
Current best tree score: -1387.946 / CPU time: 0.067
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s
--------------------------------------------------------------------
| FINALIZING TREE SEARCH |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1387.946
2. Current log-likelihood: -1387.788
3. Current log-likelihood: -1387.670
4. Current log-likelihood: -1387.581
5. Current log-likelihood: -1387.513
6. Current log-likelihood: -1387.460
Optimal log-likelihood: -1387.418
Rate parameters: A-C: 0.33477 A-G: 2.25224 A-T: 2.12637 C-G: 1.16912 C-T: 3.25669 G-T: 1.00000
Base frequencies: A: 0.243 C: 0.182 G: 0.319 T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.357
Parameters optimization took 6 rounds (0.020 sec)
BEST SCORE FOUND : -1387.418
Total tree length: 6.704
Total number of iterations: 2
CPU time used for tree search: 0.066 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.067 sec (0h:0m:0s)
Total CPU time used: 0.146 sec (0h:0m:0s)
Total wall-clock time used: 0.149 sec (0h:0m:0s)
---> SUMMARIZE RESULTS FROM 10 RUNS
Run 1 gave best log-likelihood: -1387.262
Total CPU time for 10 runs: 1.696 seconds.
Total wall-clock time for 10 runs: 1.734 seconds.
Analysis results written to:
IQ-TREE report: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1gz3ic6z/q2iqtree.iqtree
Maximum-likelihood tree: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1gz3ic6z/q2iqtree.treefile
Trees from independent runs: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1gz3ic6z/q2iqtree.runtrees
Likelihood distances: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1gz3ic6z/q2iqtree.mldist
Screen log file: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1gz3ic6z/q2iqtree.log
Date and Time: Fri Nov 1 07:41:02 2019
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.
Command: iqtree -st DNA --runs 10 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-7bbn7mkx/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1gz3ic6z/q2iqtree -nt 1 -fast
Saved Phylogeny[Unrooted] to: iqt-gtrig-fast-ms-tree.qza
Single branch tests¶
IQ-TREE provides access to a few single branch testing methods
SH-aLRT via
--p-alrt [INT >= 1000]
aBayes via
--p-abayes [TRUE | FALSE]
local bootstrap test via
--p-lbp [INT >= 1000]
Single branch tests are commonly used as an alternative to the bootstrapping
approach we’ve discussed above, as they are substantially faster and often
recommended when constructing large phylogenies (e.g. >10,000 taxa). All
three of these methods can be applied simultaneously and viewed within iTOL
as separate bootstrap support values. These values are always in listed in the
following order of alrt / lbp / abayes. We’ll go ahead and apply all of the
branch tests in our next command, while specifying the same substitution model
as above. Feel free to combine this with the --p-fast
option. 😉
qiime phylogeny iqtree \
--i-alignment masked-aligned-rep-seqs.qza \
--p-alrt 1000 \
--p-abayes \
--p-lbp 1000 \
--p-substitution-model 'GTR+I+G' \
--o-tree iqt-sbt-tree.qza \
--verbose
stdout:
IQ-TREE multicore version 1.6.12 for Mac OS X 64-bit built Aug 15 2019
Developed by Bui Quang Minh, Nguyen Lam Tung, Olga Chernomor,
Heiko Schmidt, Dominik Schrempf, Michael Woodhams.
Host: ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-2_nv7xic/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxom3yfex/q2iqtree -nt 1 -alrt 1000 -abayes -lbp 1000
Seed: 584004 (Using SPRNG - Scalable Parallel Random Number Generator)
Time: Fri Nov 1 07:41:06 2019
Kernel: AVX - 1 threads (8 CPU cores detected)
HINT: Use -nt option to specify number of threads because your CPU has 8 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.
Reading alignment file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-2_nv7xic/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
Gap/Ambiguity Composition p-value
1 e84fcf85a6a4065231dcf343bb862f1cb32abae6 40.65% passed 90.91%
2 5525fb6dab7b6577960147574465990c6df070ad 42.99% passed 99.80%
3 eb3564a35320b53cef22a77288838c7446357327 42.99% passed 25.49%
4 418f1d469f08c99976b313028cf6d3f18f61dd55 43.93% passed 71.86%
5 2e3b2c075901640c4de739473f9246385430b1ed 31.31% passed 90.76%
6 0469f8d819bd45c7638d1c8b0895270a05f34267 38.79% passed 92.82%
7 d162ed685007f5adede58f14aece31dfa1b60c18 40.65% passed 97.17%
8 1d45b2bce36cd995c5dcb755babf512e612ce8b9 41.59% passed 39.04%
9 5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8 31.31% passed 87.21%
10 206656bec2abdbc4aee37a661ef5f4a62b5dd6ae 42.99% passed 85.00%
11 606c23e79bb730ad74e3c6efd72004c36674c17a 47.20% passed 87.78%
12 682e91d7e510ab134d0625234ad224f647c14eb0 41.59% passed 31.01%
13 6a36152105590b1eb095b9503e8f1f226fc73e43 39.25% passed 86.29%
14 6ca685c39a33bfbcb3123129e7af88d573df7d6f 42.06% failed 0.02%
15 8a1c44eb462ed58b21f3fdd72dd22bb657db2980 31.78% passed 54.40%
16 9b220cae8d375ea38b8b481cb95949cda8722fcb 36.92% passed 88.78%
17 aa4698d2e2b1fa71d08e2934a923aad7374a18f6 37.85% passed 90.52%
18 b31aa3f04bc9d5e2498d45cf1983dfaf09faa258 31.78% passed 72.69%
19 d44b129a6181f052198bda3813f0802a91612441 41.59% passed 41.69%
20 ed1acad8a98e8579a44370733533ad7d3fed8006 48.13% passed 58.15%
**** TOTAL 39.77% 1 sequences failed composition chi2 test (p-value<5%; df=3)
Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
Thoroughly optimizing +I+G parameters from 10 start values...
Init pinv, alpha: 0.000, 1.000 / Estimate: 0.000, 1.237 / LogL: -1394.542
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.010, 1.344 / LogL: -1394.883
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.010, 1.353 / LogL: -1394.887
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.010, 1.352 / LogL: -1394.874
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.009, 1.349 / LogL: -1394.839
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.351 / LogL: -1394.865
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.010, 1.352 / LogL: -1394.886
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.346 / LogL: -1394.827
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.009, 1.347 / LogL: -1394.838
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.009, 1.347 / LogL: -1394.849
Optimal pinv,alpha: 0.000, 1.237 / LogL: -1394.542
Parameters optimization took 0.462 sec
Computing ML distances based on estimated model parameters... 0.003 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1392.865
--------------------------------------------------------------------
| INITIALIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.089 second
Computing log-likelihood of 98 initial trees ... 0.213 seconds
Current best score: -1392.865
Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.265
Iteration 10 / LogL: -1387.304 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.279 / Time: 0h:0m:1s
Finish initializing candidate tree set (1)
Current best tree score: -1387.265 / CPU time: 0.655
Number of iterations: 20
--------------------------------------------------------------------
| OPTIMIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Iteration 30 / LogL: -1389.882 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 40 / LogL: -1387.757 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 50 / LogL: -1388.533 / Time: 0h:0m:2s (0h:0m:2s left)
Iteration 60 / LogL: -1387.407 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 70 / LogL: -1387.616 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 80 / LogL: -1387.419 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 90 / LogL: -1387.407 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 100 / LogL: -1387.288 / Time: 0h:0m:3s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 102 ITERATIONS / Time: 0h:0m:3s
--------------------------------------------------------------------
| FINALIZING TREE SEARCH |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.265
Optimal log-likelihood: -1387.256
Rate parameters: A-C: 0.32900 A-G: 2.26254 A-T: 2.14102 C-G: 1.17611 C-T: 3.29231 G-T: 1.00000
Base frequencies: A: 0.243 C: 0.182 G: 0.319 T: 0.256
Proportion of invariable sites: 0.000
Gamma shape alpha: 1.318
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1387.256
Testing tree branches by SH-like aLRT with 1000 replicates...
Testing tree branches by local-BP test with 1000 replicates...
Testing tree branches by aBayes parametric test...
0.058 sec.
Total tree length: 6.743
Total number of iterations: 102
CPU time used for tree search: 3.193 sec (0h:0m:3s)
Wall-clock time used for tree search: 3.196 sec (0h:0m:3s)
Total CPU time used: 3.737 sec (0h:0m:3s)
Total wall-clock time used: 3.743 sec (0h:0m:3s)
Analysis results written to:
IQ-TREE report: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxom3yfex/q2iqtree.iqtree
Maximum-likelihood tree: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxom3yfex/q2iqtree.treefile
Likelihood distances: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxom3yfex/q2iqtree.mldist
Screen log file: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxom3yfex/q2iqtree.log
Date and Time: Fri Nov 1 07:41:10 2019
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-2_nv7xic/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxom3yfex/q2iqtree -nt 1 -alrt 1000 -abayes -lbp 1000
Saved Phylogeny[Unrooted] to: iqt-sbt-tree.qza
Tip
IQ-TREE search settings.
There are quite a few adjustable parameters available for iqtree
that
can be modified improve searches through “tree space” and prevent the search
algorithms from getting stuck in local optima. One particular best
practice to aid in this regard, is to adjust the following parameters:
--p-perturb-nni-strength
and --p-stop-iter
(each respectively maps
to the -pers
and -nstop
flags of iqtree
). In brief, the larger
the value for NNI (nearest-neighbor interchange) perturbation, the larger
the jumps in “tree space”. This value should be set high enough to allow the
search algorithm to avoid being trapped in local optima, but not to high
that the search is haphazardly jumping around “tree space”. That is, like
Goldilocks and the three 🐻s you need to find a setting that is “just
right”, or at least within a set of reasonable bounds. One way of assessing
this, is to do a few short trial runs using the --verbose
flag. If you
see that the likelihood values are jumping around to much, then lowering the
value for --p-perturb-nni-strength
may be warranted. As for the stopping
criteria, i.e. --p-stop-iter
, the higher this value, the more thorough
your search in “tree space”. Be aware, increasing this value may also
increase the run time. That is, the search will continue until it has
sampled a number of trees, say 100 (default), without finding a better
scoring tree. If a better tree is found, then the counter resets, and the
search continues. These two parameters deserve special consideration when a
given data set contains many short sequences, quite common for microbiome
survey data. We can modify our original command to include these extra
parameters with the recommended modifications for short sequences, i.e. a
lower value for perturbation strength (shorter reads do not contain as much
phylogenetic information, thus we should limit how far we jump around in
“tree space”) and a larger number of stop iterations. See the IQ-TREE
command reference for more details about default parameter settings.
Finally, we’ll let iqtree
perform the model testing, and automatically
determine the optimal number of CPU cores to use.
qiime phylogeny iqtree \
--i-alignment masked-aligned-rep-seqs.qza \
--p-perturb-nni-strength 0.2 \
--p-stop-iter 200 \
--p-n-cores 1 \
--o-tree iqt-nnisi-fast-tree.qza \
--verbose
stdout:
IQ-TREE multicore version 1.6.12 for Mac OS X 64-bit built Aug 15 2019
Developed by Bui Quang Minh, Nguyen Lam Tung, Olga Chernomor,
Heiko Schmidt, Dominik Schrempf, Michael Woodhams.
Host: ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-g464_3bj/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9crp_hew/q2iqtree -nt 1 -nstop 200 -pers 0.200000
Seed: 582488 (Using SPRNG - Scalable Parallel Random Number Generator)
Time: Fri Nov 1 07:41:14 2019
Kernel: AVX - 1 threads (8 CPU cores detected)
HINT: Use -nt option to specify number of threads because your CPU has 8 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.
Reading alignment file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-g464_3bj/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
Gap/Ambiguity Composition p-value
1 e84fcf85a6a4065231dcf343bb862f1cb32abae6 40.65% passed 90.91%
2 5525fb6dab7b6577960147574465990c6df070ad 42.99% passed 99.80%
3 eb3564a35320b53cef22a77288838c7446357327 42.99% passed 25.49%
4 418f1d469f08c99976b313028cf6d3f18f61dd55 43.93% passed 71.86%
5 2e3b2c075901640c4de739473f9246385430b1ed 31.31% passed 90.76%
6 0469f8d819bd45c7638d1c8b0895270a05f34267 38.79% passed 92.82%
7 d162ed685007f5adede58f14aece31dfa1b60c18 40.65% passed 97.17%
8 1d45b2bce36cd995c5dcb755babf512e612ce8b9 41.59% passed 39.04%
9 5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8 31.31% passed 87.21%
10 206656bec2abdbc4aee37a661ef5f4a62b5dd6ae 42.99% passed 85.00%
11 606c23e79bb730ad74e3c6efd72004c36674c17a 47.20% passed 87.78%
12 682e91d7e510ab134d0625234ad224f647c14eb0 41.59% passed 31.01%
13 6a36152105590b1eb095b9503e8f1f226fc73e43 39.25% passed 86.29%
14 6ca685c39a33bfbcb3123129e7af88d573df7d6f 42.06% failed 0.02%
15 8a1c44eb462ed58b21f3fdd72dd22bb657db2980 31.78% passed 54.40%
16 9b220cae8d375ea38b8b481cb95949cda8722fcb 36.92% passed 88.78%
17 aa4698d2e2b1fa71d08e2934a923aad7374a18f6 37.85% passed 90.52%
18 b31aa3f04bc9d5e2498d45cf1983dfaf09faa258 31.78% passed 72.69%
19 d44b129a6181f052198bda3813f0802a91612441 41.59% passed 41.69%
20 ed1acad8a98e8579a44370733533ad7d3fed8006 48.13% passed 58.15%
**** TOTAL 39.77% 1 sequences failed composition chi2 test (p-value<5%; df=3)
Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test 286 DNA models (sample size: 214) ...

Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1419.055
2. Current log-likelihood: -1399.481
3. Current log-likelihood: -1393.449
4. Current log-likelihood: -1392.617
5. Current log-likelihood: -1392.346
6. Current log-likelihood: -1392.194
Optimal log-likelihood: -1392.107
Rate parameters: A-C: 0.13866 A-G: 1.75800 A-T: 1.42775 C-G: 0.65275 C-T: 1.75800 G-T: 1.00000
Base frequencies: A: 0.250 C: 0.250 G: 0.250 T: 0.250
Site proportion and rates: (0.671,0.384) (0.329,2.259)
Parameters optimization took 6 rounds (0.036 sec)
Computing ML distances based on estimated model parameters... 0.002 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1389.426
--------------------------------------------------------------------
| INITIALIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.087 second
Computing log-likelihood of 98 initial trees ... 0.142 seconds
Current best score: -1389.426
Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1382.399
UPDATE BEST LOG-LIKELIHOOD: -1382.399
Iteration 10 / LogL: -1382.407 / Time: 0h:0m:0s
Iteration 20 / LogL: -1382.409 / Time: 0h:0m:0s
Finish initializing candidate tree set (1)
Current best tree score: -1382.399 / CPU time: 0.461
Number of iterations: 20
--------------------------------------------------------------------
| OPTIMIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
UPDATE BEST LOG-LIKELIHOOD: -1382.398
Iteration 30 / LogL: -1382.910 / Time: 0h:0m:0s (0h:0m:3s left)
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 32: -1382.125
Iteration 40 / LogL: -1382.899 / Time: 0h:0m:0s (0h:0m:3s left)
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 48: -1382.051
Iteration 50 / LogL: -1382.127 / Time: 0h:0m:0s (0h:0m:3s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.049
Iteration 60 / LogL: -1382.081 / Time: 0h:0m:0s (0h:0m:3s left)
Iteration 70 / LogL: -1382.125 / Time: 0h:0m:1s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.049
Iteration 80 / LogL: -1382.066 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 90 / LogL: -1382.460 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 100 / LogL: -1382.052 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 110 / LogL: -1382.151 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 120 / LogL: -1382.525 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 130 / LogL: -1382.063 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 140 / LogL: -1382.170 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 150 / LogL: -1382.465 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 160 / LogL: -1382.890 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 170 / LogL: -1384.907 / Time: 0h:0m:2s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.049
Iteration 180 / LogL: -1382.294 / Time: 0h:0m:2s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.049
Iteration 190 / LogL: -1382.049 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 200 / LogL: -1382.083 / Time: 0h:0m:2s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.049
Iteration 210 / LogL: -1382.386 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 220 / LogL: -1382.085 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 230 / LogL: -1382.049 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 240 / LogL: -1382.955 / Time: 0h:0m:2s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 249 ITERATIONS / Time: 0h:0m:2s
--------------------------------------------------------------------
| FINALIZING TREE SEARCH |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1382.049
Optimal log-likelihood: -1382.043
Rate parameters: A-C: 0.18904 A-G: 1.81946 A-T: 1.51719 C-G: 0.76290 C-T: 1.81946 G-T: 1.00000
Base frequencies: A: 0.250 C: 0.250 G: 0.250 T: 0.250
Site proportion and rates: (0.719,0.422) (0.281,2.481)
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1382.043
Total tree length: 6.894
Total number of iterations: 249
CPU time used for tree search: 2.851 sec (0h:0m:2s)
Wall-clock time used for tree search: 2.856 sec (0h:0m:2s)
Total CPU time used: 2.905 sec (0h:0m:2s)
Total wall-clock time used: 2.912 sec (0h:0m:2s)
Analysis results written to:
IQ-TREE report: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9crp_hew/q2iqtree.iqtree
Maximum-likelihood tree: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9crp_hew/q2iqtree.treefile
Likelihood distances: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9crp_hew/q2iqtree.mldist
Screen log file: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9crp_hew/q2iqtree.log
Date and Time: Fri Nov 1 07:41:18 2019
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-g464_3bj/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9crp_hew/q2iqtree -nt 1 -nstop 200 -pers 0.200000
Saved Phylogeny[Unrooted] to: iqt-nnisi-fast-tree.qza
iqtree-ultrafast-bootstrap¶
As per our discussion in the raxml-rapid-bootstrap
section above, we can
also use IQ-TREE to evaluate how well our splits / bipartitions are supported
within our phylogeny via the ultrafast bootstrap algorithm. Below, we’ll
apply the plugin’s
ultrafast bootstrap command:
automatic model selection (MFP
), perform 1000
bootstrap replicates
(minimum required), set the same generally suggested parameters for
constructing a phylogeny from short sequences, and automatically determine the
optimal number of CPU cores to use:
qiime phylogeny iqtree-ultrafast-bootstrap \
--i-alignment masked-aligned-rep-seqs.qza \
--p-perturb-nni-strength 0.2 \
--p-stop-iter 200 \
--p-n-cores 1 \
--o-tree iqt-nnisi-bootstrap-tree.qza \
--verbose
stdout:
IQ-TREE multicore version 1.6.12 for Mac OS X 64-bit built Aug 15 2019
Developed by Bui Quang Minh, Nguyen Lam Tung, Olga Chernomor,
Heiko Schmidt, Dominik Schrempf, Michael Woodhams.
Host: ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-9tle8d2n/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpnrx1h1ib/q2iqtreeufboot -nt 1 -nstop 200 -pers 0.200000
Seed: 709837 (Using SPRNG - Scalable Parallel Random Number Generator)
Time: Fri Nov 1 07:41:22 2019
Kernel: AVX - 1 threads (8 CPU cores detected)
HINT: Use -nt option to specify number of threads because your CPU has 8 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.
Reading alignment file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-9tle8d2n/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
Gap/Ambiguity Composition p-value
1 e84fcf85a6a4065231dcf343bb862f1cb32abae6 40.65% passed 90.91%
2 5525fb6dab7b6577960147574465990c6df070ad 42.99% passed 99.80%
3 eb3564a35320b53cef22a77288838c7446357327 42.99% passed 25.49%
4 418f1d469f08c99976b313028cf6d3f18f61dd55 43.93% passed 71.86%
5 2e3b2c075901640c4de739473f9246385430b1ed 31.31% passed 90.76%
6 0469f8d819bd45c7638d1c8b0895270a05f34267 38.79% passed 92.82%
7 d162ed685007f5adede58f14aece31dfa1b60c18 40.65% passed 97.17%
8 1d45b2bce36cd995c5dcb755babf512e612ce8b9 41.59% passed 39.04%
9 5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8 31.31% passed 87.21%
10 206656bec2abdbc4aee37a661ef5f4a62b5dd6ae 42.99% passed 85.00%
11 606c23e79bb730ad74e3c6efd72004c36674c17a 47.20% passed 87.78%
12 682e91d7e510ab134d0625234ad224f647c14eb0 41.59% passed 31.01%
13 6a36152105590b1eb095b9503e8f1f226fc73e43 39.25% passed 86.29%
14 6ca685c39a33bfbcb3123129e7af88d573df7d6f 42.06% failed 0.02%
15 8a1c44eb462ed58b21f3fdd72dd22bb657db2980 31.78% passed 54.40%
16 9b220cae8d375ea38b8b481cb95949cda8722fcb 36.92% passed 88.78%
17 aa4698d2e2b1fa71d08e2934a923aad7374a18f6 37.85% passed 90.52%
18 b31aa3f04bc9d5e2498d45cf1983dfaf09faa258 31.78% passed 72.69%
19 d44b129a6181f052198bda3813f0802a91612441 41.59% passed 41.69%
20 ed1acad8a98e8579a44370733533ad7d3fed8006 48.13% passed 58.15%
**** TOTAL 39.77% 1 sequences failed composition chi2 test (p-value<5%; df=3)
Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test 286 DNA models (sample size: 214) ...

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1417.970
2. Current log-likelihood: -1393.853
3. Current log-likelihood: -1392.324
4. Current log-likelihood: -1391.652
5. Current log-likelihood: -1391.390
6. Current log-likelihood: -1391.246
Optimal log-likelihood: -1391.145
Rate parameters: A-C: 0.13929 A-G: 1.83346 A-T: 1.48449 C-G: 0.74342 C-T: 1.83346 G-T: 1.00000
Base frequencies: A: 0.250 C: 0.250 G: 0.250 T: 0.250
Site proportion and rates: (0.682,0.380) (0.318,2.330)
Parameters optimization took 6 rounds (0.038 sec)
Computing ML distances based on estimated model parameters... 0.002 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1389.211
--------------------------------------------------------------------
| INITIALIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.087 second
Computing log-likelihood of 98 initial trees ... 0.137 seconds
Current best score: -1389.211
Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1382.344
Iteration 10 / LogL: -1382.351 / Time: 0h:0m:0s
Iteration 20 / LogL: -1382.354 / Time: 0h:0m:0s
Finish initializing candidate tree set (1)
Current best tree score: -1382.344 / CPU time: 0.609
Number of iterations: 20
--------------------------------------------------------------------
| OPTIMIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
UPDATE BEST LOG-LIKELIHOOD: -1382.343
UPDATE BEST LOG-LIKELIHOOD: -1382.343
UPDATE BEST LOG-LIKELIHOOD: -1382.343
Iteration 30 / LogL: -1383.049 / Time: 0h:0m:0s (0h:0m:5s left)
Iteration 40 / LogL: -1382.871 / Time: 0h:0m:1s (0h:0m:4s left)
Iteration 50 / LogL: -1382.907 / Time: 0h:0m:1s (0h:0m:3s left)
Log-likelihood cutoff on original alignment: -1414.398
Iteration 60 / LogL: -1382.877 / Time: 0h:0m:1s (0h:0m:3s left)
Estimate model parameters (epsilon = 0.100)
UPDATE BEST LOG-LIKELIHOOD: -1382.098
Iteration 70 / LogL: -1382.915 / Time: 0h:0m:1s (0h:0m:3s left)
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 71: -1382.026
UPDATE BEST LOG-LIKELIHOOD: -1382.019
UPDATE BEST LOG-LIKELIHOOD: -1382.019
Iteration 80 / LogL: -1382.097 / Time: 0h:0m:1s (0h:0m:4s left)
Iteration 90 / LogL: -1382.424 / Time: 0h:0m:2s (0h:0m:4s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.019
Iteration 100 / LogL: -1382.020 / Time: 0h:0m:2s (0h:0m:3s left)
Log-likelihood cutoff on original alignment: -1415.123
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.976
NOTE: UFBoot does not converge, continue at least 100 more iterations
Iteration 110 / LogL: -1382.875 / Time: 0h:0m:2s (0h:0m:3s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.018
Iteration 120 / LogL: -1382.018 / Time: 0h:0m:2s (0h:0m:3s left)
Iteration 130 / LogL: -1382.440 / Time: 0h:0m:2s (0h:0m:3s left)
Iteration 140 / LogL: -1382.441 / Time: 0h:0m:3s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.018
Iteration 150 / LogL: -1382.423 / Time: 0h:0m:3s (0h:0m:2s left)
Log-likelihood cutoff on original alignment: -1415.123
Iteration 160 / LogL: -1382.065 / Time: 0h:0m:3s (0h:0m:2s left)
Iteration 170 / LogL: -1382.100 / Time: 0h:0m:3s (0h:0m:2s left)
Iteration 180 / LogL: -1382.646 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 190 / LogL: -1382.103 / Time: 0h:0m:4s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.018
Iteration 200 / LogL: -1382.019 / Time: 0h:0m:4s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1415.806
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.997
Iteration 210 / LogL: -1382.030 / Time: 0h:0m:4s (0h:0m:1s left)
Iteration 220 / LogL: -1382.031 / Time: 0h:0m:4s (0h:0m:1s left)
Iteration 230 / LogL: -1382.031 / Time: 0h:0m:4s (0h:0m:1s left)
Iteration 240 / LogL: -1382.096 / Time: 0h:0m:5s (0h:0m:1s left)
Iteration 250 / LogL: -1382.019 / Time: 0h:0m:5s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1415.806
Iteration 260 / LogL: -1385.409 / Time: 0h:0m:5s (0h:0m:0s left)
Iteration 270 / LogL: -1382.044 / Time: 0h:0m:5s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 272 ITERATIONS / Time: 0h:0m:5s
--------------------------------------------------------------------
| FINALIZING TREE SEARCH |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1382.018
Optimal log-likelihood: -1382.012
Rate parameters: A-C: 0.18912 A-G: 1.82226 A-T: 1.51736 C-G: 0.76287 C-T: 1.82226 G-T: 1.00000
Base frequencies: A: 0.250 C: 0.250 G: 0.250 T: 0.250
Site proportion and rates: (0.721,0.413) (0.279,2.515)
Parameters optimization took 1 rounds (0.005 sec)
BEST SCORE FOUND : -1382.012
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpnrx1h1ib/q2iqtreeufboot.splits.nex
Total tree length: 7.029
Total number of iterations: 272
CPU time used for tree search: 5.817 sec (0h:0m:5s)
Wall-clock time used for tree search: 5.833 sec (0h:0m:5s)
Total CPU time used: 5.954 sec (0h:0m:5s)
Total wall-clock time used: 5.975 sec (0h:0m:5s)
Computing bootstrap consensus tree...
Reading input file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpnrx1h1ib/q2iqtreeufboot.splits.nex...
20 taxa and 145 splits.
Consensus tree written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpnrx1h1ib/q2iqtreeufboot.contree
Reading input trees file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpnrx1h1ib/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1382.012
Analysis results written to:
IQ-TREE report: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpnrx1h1ib/q2iqtreeufboot.iqtree
Maximum-likelihood tree: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpnrx1h1ib/q2iqtreeufboot.treefile
Likelihood distances: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpnrx1h1ib/q2iqtreeufboot.mldist
Ultrafast bootstrap approximation results written to:
Split support values: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpnrx1h1ib/q2iqtreeufboot.splits.nex
Consensus tree: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpnrx1h1ib/q2iqtreeufboot.contree
Screen log file: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpnrx1h1ib/q2iqtreeufboot.log
Date and Time: Fri Nov 1 07:41:29 2019
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.
Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-9tle8d2n/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpnrx1h1ib/q2iqtreeufboot -nt 1 -nstop 200 -pers 0.200000
Saved Phylogeny[Unrooted] to: iqt-nnisi-bootstrap-tree.qza
Perform single branch tests alongside ufboot¶
We can also apply single branch test methods concurrently with ultrafast bootstrapping. The support values will always be represented in the following order: alrt / lbp / abayes / ufboot. Again, these values can be seen as separately listed bootstrap values in iTOL. We’ll also specify a model as we did earlier.
qiime phylogeny iqtree-ultrafast-bootstrap \
--i-alignment masked-aligned-rep-seqs.qza \
--p-perturb-nni-strength 0.2 \
--p-stop-iter 200 \
--p-n-cores 1 \
--p-alrt 1000 \
--p-abayes \
--p-lbp 1000 \
--p-substitution-model 'GTR+I+G' \
--o-tree iqt-nnisi-bootstrap-sbt-gtrig-tree.qza \
--verbose
stdout:
IQ-TREE multicore version 1.6.12 for Mac OS X 64-bit built Aug 15 2019
Developed by Bui Quang Minh, Nguyen Lam Tung, Olga Chernomor,
Heiko Schmidt, Dominik Schrempf, Michael Woodhams.
Host: ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-hekktd9a/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwbblgu6y/q2iqtreeufboot -nt 1 -alrt 1000 -abayes -lbp 1000 -nstop 200 -pers 0.200000
Seed: 960019 (Using SPRNG - Scalable Parallel Random Number Generator)
Time: Fri Nov 1 07:41:33 2019
Kernel: AVX - 1 threads (8 CPU cores detected)
HINT: Use -nt option to specify number of threads because your CPU has 8 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.
Reading alignment file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-hekktd9a/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
Gap/Ambiguity Composition p-value
1 e84fcf85a6a4065231dcf343bb862f1cb32abae6 40.65% passed 90.91%
2 5525fb6dab7b6577960147574465990c6df070ad 42.99% passed 99.80%
3 eb3564a35320b53cef22a77288838c7446357327 42.99% passed 25.49%
4 418f1d469f08c99976b313028cf6d3f18f61dd55 43.93% passed 71.86%
5 2e3b2c075901640c4de739473f9246385430b1ed 31.31% passed 90.76%
6 0469f8d819bd45c7638d1c8b0895270a05f34267 38.79% passed 92.82%
7 d162ed685007f5adede58f14aece31dfa1b60c18 40.65% passed 97.17%
8 1d45b2bce36cd995c5dcb755babf512e612ce8b9 41.59% passed 39.04%
9 5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8 31.31% passed 87.21%
10 206656bec2abdbc4aee37a661ef5f4a62b5dd6ae 42.99% passed 85.00%
11 606c23e79bb730ad74e3c6efd72004c36674c17a 47.20% passed 87.78%
12 682e91d7e510ab134d0625234ad224f647c14eb0 41.59% passed 31.01%
13 6a36152105590b1eb095b9503e8f1f226fc73e43 39.25% passed 86.29%
14 6ca685c39a33bfbcb3123129e7af88d573df7d6f 42.06% failed 0.02%
15 8a1c44eb462ed58b21f3fdd72dd22bb657db2980 31.78% passed 54.40%
16 9b220cae8d375ea38b8b481cb95949cda8722fcb 36.92% passed 88.78%
17 aa4698d2e2b1fa71d08e2934a923aad7374a18f6 37.85% passed 90.52%
18 b31aa3f04bc9d5e2498d45cf1983dfaf09faa258 31.78% passed 72.69%
19 d44b129a6181f052198bda3813f0802a91612441 41.59% passed 41.69%
20 ed1acad8a98e8579a44370733533ad7d3fed8006 48.13% passed 58.15%
**** TOTAL 39.77% 1 sequences failed composition chi2 test (p-value<5%; df=3)
Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
Generating 1000 samples for ultrafast bootstrap (seed: 960019)...
NOTE: 1 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
Thoroughly optimizing +I+G parameters from 10 start values...
Init pinv, alpha: 0.000, 1.000 / Estimate: 0.000, 1.237 / LogL: -1394.542
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.010, 1.344 / LogL: -1394.883
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.010, 1.353 / LogL: -1394.887
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.010, 1.352 / LogL: -1394.874
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.009, 1.348 / LogL: -1394.840
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.351 / LogL: -1394.865
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.010, 1.352 / LogL: -1394.886
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.346 / LogL: -1394.827
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.009, 1.347 / LogL: -1394.838
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.009, 1.347 / LogL: -1394.849
Optimal pinv,alpha: 0.000, 1.237 / LogL: -1394.542
Parameters optimization took 0.465 sec
Computing ML distances based on estimated model parameters... 0.003 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1392.865
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| INITIALIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.084 second
Computing log-likelihood of 98 initial trees ... 0.200 seconds
Current best score: -1392.865
Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.265
UPDATE BEST LOG-LIKELIHOOD: -1387.265
Iteration 10 / LogL: -1387.265 / Time: 0h:0m:1s
Iteration 20 / LogL: -1387.279 / Time: 0h:0m:1s
Finish initializing candidate tree set (1)
Current best tree score: -1387.265 / CPU time: 0.799
Number of iterations: 20
--------------------------------------------------------------------
| OPTIMIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
UPDATE BEST LOG-LIKELIHOOD: -1387.259
UPDATE BEST LOG-LIKELIHOOD: -1387.257
Iteration 30 / LogL: -1387.347 / Time: 0h:0m:1s (0h:0m:9s left)
Iteration 40 / LogL: -1387.332 / Time: 0h:0m:1s (0h:0m:7s left)
Iteration 50 / LogL: -1387.414 / Time: 0h:0m:1s (0h:0m:6s left)
Log-likelihood cutoff on original alignment: -1405.359
Iteration 60 / LogL: -1387.347 / Time: 0h:0m:2s (0h:0m:5s left)
Iteration 70 / LogL: -1387.347 / Time: 0h:0m:2s (0h:0m:5s left)
Iteration 80 / LogL: -1387.348 / Time: 0h:0m:3s (0h:0m:4s left)
Iteration 90 / LogL: -1387.347 / Time: 0h:0m:3s (0h:0m:4s left)
Iteration 100 / LogL: -1389.337 / Time: 0h:0m:3s (0h:0m:3s left)
Log-likelihood cutoff on original alignment: -1406.775
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.998
Iteration 110 / LogL: -1387.347 / Time: 0h:0m:3s (0h:0m:3s left)
Iteration 120 / LogL: -1387.614 / Time: 0h:0m:4s (0h:0m:2s left)
Iteration 130 / LogL: -1387.347 / Time: 0h:0m:4s (0h:0m:2s left)
Iteration 140 / LogL: -1387.446 / Time: 0h:0m:4s (0h:0m:2s left)
Iteration 150 / LogL: -1387.543 / Time: 0h:0m:5s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1406.775
Iteration 160 / LogL: -1387.347 / Time: 0h:0m:5s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.256
Iteration 170 / LogL: -1387.349 / Time: 0h:0m:5s (0h:0m:1s left)
Iteration 180 / LogL: -1387.347 / Time: 0h:0m:6s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.256
Iteration 190 / LogL: -1387.415 / Time: 0h:0m:6s (0h:0m:0s left)
Iteration 200 / LogL: -1387.361 / Time: 0h:0m:6s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1406.708
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.998
TREE SEARCH COMPLETED AFTER 202 ITERATIONS / Time: 0h:0m:6s
--------------------------------------------------------------------
| FINALIZING TREE SEARCH |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.256
Optimal log-likelihood: -1387.253
Rate parameters: A-C: 0.32957 A-G: 2.26332 A-T: 2.14163 C-G: 1.17419 C-T: 3.29400 G-T: 1.00000
Base frequencies: A: 0.243 C: 0.182 G: 0.319 T: 0.256
Proportion of invariable sites: 0.000
Gamma shape alpha: 1.318
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1387.253
Testing tree branches by SH-like aLRT with 1000 replicates...
Testing tree branches by local-BP test with 1000 replicates...
Testing tree branches by aBayes parametric test...
0.055 sec.
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwbblgu6y/q2iqtreeufboot.splits.nex
Total tree length: 6.746
Total number of iterations: 202
CPU time used for tree search: 6.472 sec (0h:0m:6s)
Wall-clock time used for tree search: 6.486 sec (0h:0m:6s)
Total CPU time used: 7.094 sec (0h:0m:7s)
Total wall-clock time used: 7.114 sec (0h:0m:7s)
Computing bootstrap consensus tree...
Reading input file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwbblgu6y/q2iqtreeufboot.splits.nex...
20 taxa and 147 splits.
Consensus tree written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwbblgu6y/q2iqtreeufboot.contree
Reading input trees file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwbblgu6y/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1387.430
Analysis results written to:
IQ-TREE report: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwbblgu6y/q2iqtreeufboot.iqtree
Maximum-likelihood tree: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwbblgu6y/q2iqtreeufboot.treefile
Likelihood distances: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwbblgu6y/q2iqtreeufboot.mldist
Ultrafast bootstrap approximation results written to:
Split support values: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwbblgu6y/q2iqtreeufboot.splits.nex
Consensus tree: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwbblgu6y/q2iqtreeufboot.contree
Screen log file: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwbblgu6y/q2iqtreeufboot.log
Date and Time: Fri Nov 1 07:41:41 2019
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.
Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-hekktd9a/f3132e20-be7c-4b66-9769-15edeb3b3842/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwbblgu6y/q2iqtreeufboot -nt 1 -alrt 1000 -abayes -lbp 1000 -nstop 200 -pers 0.200000
Saved Phylogeny[Unrooted] to: iqt-nnisi-bootstrap-sbt-gtrig-tree.qza
Tip
If there is a need to reduce the impact of potential model
violations that occur during a UFBoot search, and / or would simply
like to be more rigorous, we can add the --p-bnni
option to any of the
iqtree-ultrafast-bootstrap
commands above.
Root the phylogeny¶
In order to make proper use of diversity metrics such as UniFrac, the phylogeny must be rooted. Typically an outgroup is chosen when rooting a tree. In general, phylogenetic inference tools using Maximum Likelihood often return an unrooted tree by default.
QIIME 2 provides a way to
mid-point root our
phylogeny. Other rooting options may be available in the future. For now, we’ll
root our bootstrap tree from iqtree-ultrafast-bootstrap
like so:
qiime phylogeny midpoint-root \
--i-tree iqt-nnisi-bootstrap-sbt-gtrig-tree.qza \
--o-rooted-tree iqt-nnisi-bootstrap-sbt-gtrig-tree-rooted.qza
Tip
iTOL viewing Reminder. We can view our tree and its associated alignment via iTOL. All you need to do is upload the iqt-nnisi-bootstrap-sbt-gtrig-tree-rooted.qza tree file. Display the tree in Normal mode. Then drag and drop the masked-aligned-rep-seqs.qza file onto the visualization. Now you can view the phylogeny alongside the alignment.
Pipelines¶
Here we will outline the use of the phylogeny pipeline align-to-tree-mafft-fasttree
One advantage of pipelines is that they combine ordered sets of commonly used commands, into one condensed simple command. To keep these “convenience” pipelines easy to use, it is quite common to only expose a few options to the user. That is, most of the commands executed via pipelines are often configured to use default option settings. However, options that are deemed important enough for the user to consider setting, are made available. The options exposed via a given pipeline will largely depend upon what it is doing. Pipelines are also a great way for new users to get started, as it helps to lay a foundation of good practices in setting up standard operating procedures.
Rather than run one or more of the following QIIME 2 commands listed below:
qiime alignment mafft ...
qiime alignment mask ...
qiime phylogeny fasttree ...
qiime phylogeny midpoint-root ...
We can make use of the pipeline align-to-tree-mafft-fasttree to automate the above four steps in one go. Here is the description taken from the pipeline help doc:
This pipeline will start by creating a sequence alignment using MAFFT, after which any alignment columns that are phylogenetically uninformative or ambiguously aligned will be removed (masked). The resulting masked alignment will be used to infer a phylogenetic tree and then subsequently rooted at its midpoint. Output files from each step of the pipeline will be saved. This includes both the unmasked and masked MAFFT alignment from q2-alignment methods, and both the rooted and unrooted phylogenies from q2-phylogeny methods.
This can all be accomplished by simply running the following:
qiime phylogeny align-to-tree-mafft-fasttree \
--i-sequences rep-seqs.qza \
--output-dir mafft-fasttree-output
Output artifacts:
Congratulations! You now know how to construct a phylogeny in QIIME 2!