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/2020.2/tutorials/phylogeny/rep-seqs.qza
Save as: rep-seqs.qza
wget \
-O "rep-seqs.qza" \
"https://data.qiime2.org/2020.2/tutorials/phylogeny/rep-seqs.qza"
curl -sL \
"https://data.qiime2.org/2020.2/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 6415 -N 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-wm3gc8d3/c67b6543-8e61-435d-b61e-36a863284e6e/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp4dzvt6iy -n q2
Partition: 0 with name: No Name Provided
Base frequencies: 0.243 0.182 0.319 0.256
Inference[0]: Time 0.651053 CAT-based likelihood -1242.579881, best rearrangement setting 5
Conducting final model optimizations on all 1 trees under GAMMA-based models ....
Inference[0] final GAMMA-based Likelihood: -1388.335399 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp4dzvt6iy/RAxML_result.q2
Starting final GAMMA-based thorough Optimization on tree 0 likelihood -1388.335399 ....
Final GAMMA-based Score of best tree -1387.278976
Program execution info written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp4dzvt6iy/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp4dzvt6iy/RAxML_bestTree.q2
Overall execution time: 1.321623 secs or 0.000367 hours or 0.000015 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 6415 -N 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-wm3gc8d3/c67b6543-8e61-435d-b61e-36a863284e6e/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp4dzvt6iy -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-k2e04py8/c67b6543-8e61-435d-b61e-36a863284e6e/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpdiga50gu -n q2
Partition: 0 with name: No Name Provided
Base frequencies: 0.243 0.182 0.319 0.256
Inference[0]: Time 0.623663 CAT-based likelihood -1238.242991, best rearrangement setting 5
Inference[1]: Time 0.509429 CAT-based likelihood -1249.502284, best rearrangement setting 5
Inference[2]: Time 0.524310 CAT-based likelihood -1242.978035, best rearrangement setting 5
Inference[3]: Time 0.666095 CAT-based likelihood -1243.159855, best rearrangement setting 5
Inference[4]: Time 0.519867 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/tmpdiga50gu/RAxML_result.q2.RUN.0
Inference[1] final GAMMA-based Likelihood: -1392.813982 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpdiga50gu/RAxML_result.q2.RUN.1
Inference[2] final GAMMA-based Likelihood: -1388.073642 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpdiga50gu/RAxML_result.q2.RUN.2
Inference[3] final GAMMA-based Likelihood: -1387.945266 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpdiga50gu/RAxML_result.q2.RUN.3
Inference[4] final GAMMA-based Likelihood: -1387.557031 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpdiga50gu/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/tmpdiga50gu/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpdiga50gu/RAxML_bestTree.q2
Overall execution time: 3.624212 secs or 0.001007 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-k2e04py8/c67b6543-8e61-435d-b61e-36a863284e6e/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpdiga50gu -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-ayhruhog/c67b6543-8e61-435d-b61e-36a863284e6e/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpcgeh77_f -n q2bootstrap
Time for BS model parameter optimization 0.052080
Bootstrap[0]: Time 0.181618 seconds, bootstrap likelihood -1199.758796, best rearrangement setting 12
Bootstrap[1]: Time 0.120677 seconds, bootstrap likelihood -1344.229251, best rearrangement setting 6
Bootstrap[2]: Time 0.108588 seconds, bootstrap likelihood -1295.343000, best rearrangement setting 8
Bootstrap[3]: Time 0.093386 seconds, bootstrap likelihood -1273.768320, best rearrangement setting 8
Bootstrap[4]: Time 0.108861 seconds, bootstrap likelihood -1253.402952, best rearrangement setting 6
Bootstrap[5]: Time 0.126696 seconds, bootstrap likelihood -1260.866113, best rearrangement setting 10
Bootstrap[6]: Time 0.112777 seconds, bootstrap likelihood -1293.636299, best rearrangement setting 14
Bootstrap[7]: Time 0.106289 seconds, bootstrap likelihood -1227.178693, best rearrangement setting 6
Bootstrap[8]: Time 0.110966 seconds, bootstrap likelihood -1321.820787, best rearrangement setting 13
Bootstrap[9]: Time 0.121619 seconds, bootstrap likelihood -1147.233446, best rearrangement setting 6
Bootstrap[10]: Time 0.084652 seconds, bootstrap likelihood -1220.766493, best rearrangement setting 13
Bootstrap[11]: Time 0.120440 seconds, bootstrap likelihood -1200.006355, best rearrangement setting 8
Bootstrap[12]: Time 0.129327 seconds, bootstrap likelihood -1346.392834, best rearrangement setting 14
Bootstrap[13]: Time 0.111474 seconds, bootstrap likelihood -1301.111096, best rearrangement setting 14
Bootstrap[14]: Time 0.118893 seconds, bootstrap likelihood -1262.253559, best rearrangement setting 11
Bootstrap[15]: Time 0.116589 seconds, bootstrap likelihood -1215.017551, best rearrangement setting 14
Bootstrap[16]: Time 0.110033 seconds, bootstrap likelihood -1238.832009, best rearrangement setting 7
Bootstrap[17]: Time 0.096895 seconds, bootstrap likelihood -1393.989732, best rearrangement setting 12
Bootstrap[18]: Time 0.108248 seconds, bootstrap likelihood -1173.921002, best rearrangement setting 15
Bootstrap[19]: Time 0.108892 seconds, bootstrap likelihood -1185.726976, best rearrangement setting 11
Bootstrap[20]: Time 0.100850 seconds, bootstrap likelihood -1158.491940, best rearrangement setting 6
Bootstrap[21]: Time 0.093500 seconds, bootstrap likelihood -1154.664272, best rearrangement setting 11
Bootstrap[22]: Time 0.104642 seconds, bootstrap likelihood -1244.159837, best rearrangement setting 10
Bootstrap[23]: Time 0.127629 seconds, bootstrap likelihood -1211.171036, best rearrangement setting 15
Bootstrap[24]: Time 0.101407 seconds, bootstrap likelihood -1261.440677, best rearrangement setting 12
Bootstrap[25]: Time 0.107081 seconds, bootstrap likelihood -1331.836715, best rearrangement setting 15
Bootstrap[26]: Time 0.110434 seconds, bootstrap likelihood -1129.144509, best rearrangement setting 5
Bootstrap[27]: Time 0.133349 seconds, bootstrap likelihood -1226.624056, best rearrangement setting 7
Bootstrap[28]: Time 0.135498 seconds, bootstrap likelihood -1221.046176, best rearrangement setting 12
Bootstrap[29]: Time 0.091955 seconds, bootstrap likelihood -1211.791204, best rearrangement setting 14
Bootstrap[30]: Time 0.114907 seconds, bootstrap likelihood -1389.442380, best rearrangement setting 5
Bootstrap[31]: Time 0.112914 seconds, bootstrap likelihood -1303.638592, best rearrangement setting 12
Bootstrap[32]: Time 0.124271 seconds, bootstrap likelihood -1172.859456, best rearrangement setting 12
Bootstrap[33]: Time 0.106678 seconds, bootstrap likelihood -1244.617135, best rearrangement setting 9
Bootstrap[34]: Time 0.101719 seconds, bootstrap likelihood -1211.871717, best rearrangement setting 15
Bootstrap[35]: Time 0.123494 seconds, bootstrap likelihood -1299.862912, best rearrangement setting 5
Bootstrap[36]: Time 0.098445 seconds, bootstrap likelihood -1141.967505, best rearrangement setting 5
Bootstrap[37]: Time 0.121983 seconds, bootstrap likelihood -1283.923198, best rearrangement setting 12
Bootstrap[38]: Time 0.102102 seconds, bootstrap likelihood -1304.250946, best rearrangement setting 5
Bootstrap[39]: Time 0.093806 seconds, bootstrap likelihood -1407.084376, best rearrangement setting 15
Bootstrap[40]: Time 0.114389 seconds, bootstrap likelihood -1277.946299, best rearrangement setting 13
Bootstrap[41]: Time 0.113969 seconds, bootstrap likelihood -1279.006200, best rearrangement setting 7
Bootstrap[42]: Time 0.105849 seconds, bootstrap likelihood -1160.274606, best rearrangement setting 6
Bootstrap[43]: Time 0.128212 seconds, bootstrap likelihood -1216.079259, best rearrangement setting 14
Bootstrap[44]: Time 0.098106 seconds, bootstrap likelihood -1382.278311, best rearrangement setting 8
Bootstrap[45]: Time 0.115715 seconds, bootstrap likelihood -1099.004439, best rearrangement setting 11
Bootstrap[46]: Time 0.093864 seconds, bootstrap likelihood -1296.527478, best rearrangement setting 8
Bootstrap[47]: Time 0.131588 seconds, bootstrap likelihood -1291.322658, best rearrangement setting 9
Bootstrap[48]: Time 0.088062 seconds, bootstrap likelihood -1161.908080, best rearrangement setting 6
Bootstrap[49]: Time 0.121469 seconds, bootstrap likelihood -1257.348428, best rearrangement setting 13
Bootstrap[50]: Time 0.143257 seconds, bootstrap likelihood -1309.422533, best rearrangement setting 13
Bootstrap[51]: Time 0.098795 seconds, bootstrap likelihood -1197.633097, best rearrangement setting 11
Bootstrap[52]: Time 0.112454 seconds, bootstrap likelihood -1347.123005, best rearrangement setting 8
Bootstrap[53]: Time 0.103337 seconds, bootstrap likelihood -1234.934890, best rearrangement setting 14
Bootstrap[54]: Time 0.115260 seconds, bootstrap likelihood -1227.092434, best rearrangement setting 6
Bootstrap[55]: Time 0.118732 seconds, bootstrap likelihood -1280.635747, best rearrangement setting 7
Bootstrap[56]: Time 0.099194 seconds, bootstrap likelihood -1225.911449, best rearrangement setting 6
Bootstrap[57]: Time 0.101892 seconds, bootstrap likelihood -1236.213347, best rearrangement setting 11
Bootstrap[58]: Time 0.138348 seconds, bootstrap likelihood -1393.245723, best rearrangement setting 14
Bootstrap[59]: Time 0.126303 seconds, bootstrap likelihood -1212.039371, best rearrangement setting 6
Bootstrap[60]: Time 0.097597 seconds, bootstrap likelihood -1248.692011, best rearrangement setting 10
Bootstrap[61]: Time 0.113407 seconds, bootstrap likelihood -1172.820979, best rearrangement setting 13
Bootstrap[62]: Time 0.125327 seconds, bootstrap likelihood -1126.745788, best rearrangement setting 14
Bootstrap[63]: Time 0.103499 seconds, bootstrap likelihood -1267.434444, best rearrangement setting 12
Bootstrap[64]: Time 0.095928 seconds, bootstrap likelihood -1340.680748, best rearrangement setting 5
Bootstrap[65]: Time 0.101131 seconds, bootstrap likelihood -1072.671059, best rearrangement setting 5
Bootstrap[66]: Time 0.123986 seconds, bootstrap likelihood -1234.294838, best rearrangement setting 8
Bootstrap[67]: Time 0.123783 seconds, bootstrap likelihood -1109.249439, best rearrangement setting 15
Bootstrap[68]: Time 0.095088 seconds, bootstrap likelihood -1314.493588, best rearrangement setting 8
Bootstrap[69]: Time 0.098507 seconds, bootstrap likelihood -1173.850035, best rearrangement setting 13
Bootstrap[70]: Time 0.123258 seconds, bootstrap likelihood -1231.066465, best rearrangement setting 10
Bootstrap[71]: Time 0.103306 seconds, bootstrap likelihood -1146.861379, best rearrangement setting 9
Bootstrap[72]: Time 0.091666 seconds, bootstrap likelihood -1148.753369, best rearrangement setting 8
Bootstrap[73]: Time 0.105392 seconds, bootstrap likelihood -1333.374056, best rearrangement setting 9
Bootstrap[74]: Time 0.089935 seconds, bootstrap likelihood -1259.382378, best rearrangement setting 5
Bootstrap[75]: Time 0.097522 seconds, bootstrap likelihood -1319.944496, best rearrangement setting 6
Bootstrap[76]: Time 0.110489 seconds, bootstrap likelihood -1309.042165, best rearrangement setting 14
Bootstrap[77]: Time 0.133879 seconds, bootstrap likelihood -1232.061289, best rearrangement setting 8
Bootstrap[78]: Time 0.110360 seconds, bootstrap likelihood -1261.333984, best rearrangement setting 9
Bootstrap[79]: Time 0.113974 seconds, bootstrap likelihood -1194.644341, best rearrangement setting 13
Bootstrap[80]: Time 0.100860 seconds, bootstrap likelihood -1214.037389, best rearrangement setting 9
Bootstrap[81]: Time 0.117326 seconds, bootstrap likelihood -1224.527657, best rearrangement setting 8
Bootstrap[82]: Time 0.135541 seconds, bootstrap likelihood -1241.464826, best rearrangement setting 11
Bootstrap[83]: Time 0.097520 seconds, bootstrap likelihood -1230.730558, best rearrangement setting 6
Bootstrap[84]: Time 0.108422 seconds, bootstrap likelihood -1219.034592, best rearrangement setting 10
Bootstrap[85]: Time 0.110529 seconds, bootstrap likelihood -1280.071994, best rearrangement setting 8
Bootstrap[86]: Time 0.098919 seconds, bootstrap likelihood -1444.747777, best rearrangement setting 9
Bootstrap[87]: Time 0.096856 seconds, bootstrap likelihood -1245.890035, best rearrangement setting 14
Bootstrap[88]: Time 0.110396 seconds, bootstrap likelihood -1287.832766, best rearrangement setting 7
Bootstrap[89]: Time 0.105908 seconds, bootstrap likelihood -1325.245976, best rearrangement setting 5
Bootstrap[90]: Time 0.120951 seconds, bootstrap likelihood -1227.883697, best rearrangement setting 5
Bootstrap[91]: Time 0.111035 seconds, bootstrap likelihood -1273.489392, best rearrangement setting 8
Bootstrap[92]: Time 0.046177 seconds, bootstrap likelihood -1234.725870, best rearrangement setting 7
Bootstrap[93]: Time 0.118856 seconds, bootstrap likelihood -1235.733064, best rearrangement setting 11
Bootstrap[94]: Time 0.097637 seconds, bootstrap likelihood -1204.319488, best rearrangement setting 15
Bootstrap[95]: Time 0.095236 seconds, bootstrap likelihood -1183.328582, best rearrangement setting 11
Bootstrap[96]: Time 0.110232 seconds, bootstrap likelihood -1196.298898, best rearrangement setting 13
Bootstrap[97]: Time 0.116684 seconds, bootstrap likelihood -1339.251746, best rearrangement setting 12
Bootstrap[98]: Time 0.045519 seconds, bootstrap likelihood -1404.363552, best rearrangement setting 7
Bootstrap[99]: Time 0.059553 seconds, bootstrap likelihood -1270.157811, best rearrangement setting 7
Overall Time for 100 Rapid Bootstraps 10.947528 seconds
Average Time per Rapid Bootstrap 0.109475 seconds
Starting ML Search ...
Fast ML optimization finished
Fast ML search Time: 4.379153 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: 2.217905 seconds
Thorough ML search Time: 0.593935 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 7.197641 secs or 0.001999 hours
Combined Bootstrap and ML search took 18.145492 secs or 0.005040 hours
Drawing Bootstrap Support Values on best-scoring ML tree ...
Found 1 tree in File /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpcgeh77_f/RAxML_bestTree.q2bootstrap
Found 1 tree in File /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpcgeh77_f/RAxML_bestTree.q2bootstrap
Program execution info written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpcgeh77_f/RAxML_info.q2bootstrap
All 100 bootstrapped trees written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpcgeh77_f/RAxML_bootstrap.q2bootstrap
Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpcgeh77_f/RAxML_bestTree.q2bootstrap
Best-scoring ML tree with support values written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpcgeh77_f/RAxML_bipartitions.q2bootstrap
Best-scoring ML tree with support values as branch labels written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpcgeh77_f/RAxML_bipartitionsBranchLabels.q2bootstrap
Overall execution time for full ML analysis: 18.158612 secs or 0.005044 hours or 0.000210 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-ayhruhog/c67b6543-8e61-435d-b61e-36a863284e6e/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpcgeh77_f -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-00veqgb9/c67b6543-8e61-435d-b61e-36a863284e6e/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpb4ro6vaj/q2iqtree -nt 1
Seed: 733653 (Using SPRNG - Scalable Parallel Random Number Generator)
Time: Fri Feb 28 11:27:37 2020
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-00veqgb9/c67b6543-8e61-435d-b61e-36a863284e6e/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: -1418.019
2. Current log-likelihood: -1393.889
3. Current log-likelihood: -1392.403
4. Current log-likelihood: -1391.726
5. Current log-likelihood: -1391.446
6. Current log-likelihood: -1391.309
Optimal log-likelihood: -1391.208
Rate parameters: A-C: 0.13750 A-G: 1.82207 A-T: 1.46648 C-G: 0.74131 C-T: 1.82207 G-T: 1.00000
Base frequencies: A: 0.250 C: 0.250 G: 0.250 T: 0.250
Site proportion and rates: (0.681,0.380) (0.319,2.325)
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: -1389.219
--------------------------------------------------------------------
| INITIALIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.088 second
Computing log-likelihood of 98 initial trees ... 0.143 seconds
Current best score: -1389.219
Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1382.347
Iteration 10 / LogL: -1382.348 / Time: 0h:0m:0s
Iteration 20 / LogL: -1382.348 / Time: 0h:0m:0s
Finish initializing candidate tree set (1)
Current best tree score: -1382.347 / CPU time: 0.469
Number of iterations: 20
--------------------------------------------------------------------
| OPTIMIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Iteration 30 / LogL: -1382.350 / Time: 0h:0m:0s (0h:0m:1s left)
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 38: -1382.104
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 39: -1382.022
Iteration 40 / LogL: -1382.095 / Time: 0h:0m:0s (0h:0m:2s left)
Iteration 50 / LogL: -1403.480 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 60 / LogL: -1382.082 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 70 / LogL: -1391.218 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 80 / LogL: -1382.099 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 90 / LogL: -1382.096 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 100 / LogL: -1382.307 / Time: 0h:0m:1s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.020
Iteration 110 / LogL: -1382.105 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 120 / LogL: -1382.036 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 130 / LogL: -1382.866 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 140 / LogL: -1382.031 / Time: 0h:0m:2s (0h:0m:-1s left)
TREE SEARCH COMPLETED AFTER 140 ITERATIONS / Time: 0h:0m:2s
--------------------------------------------------------------------
| FINALIZING TREE SEARCH |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1382.020
Optimal log-likelihood: -1382.014
Rate parameters: A-C: 0.19304 A-G: 1.84742 A-T: 1.54181 C-G: 0.77458 C-T: 1.84742 G-T: 1.00000
Base frequencies: A: 0.250 C: 0.250 G: 0.250 T: 0.250
Site proportion and rates: (0.720,0.413) (0.280,2.513)
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1382.014
Total tree length: 7.031
Total number of iterations: 140
CPU time used for tree search: 2.336 sec (0h:0m:2s)
Wall-clock time used for tree search: 2.345 sec (0h:0m:2s)
Total CPU time used: 2.390 sec (0h:0m:2s)
Total wall-clock time used: 2.402 sec (0h:0m:2s)
Analysis results written to:
IQ-TREE report: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpb4ro6vaj/q2iqtree.iqtree
Maximum-likelihood tree: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpb4ro6vaj/q2iqtree.treefile
Likelihood distances: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpb4ro6vaj/q2iqtree.mldist
Screen log file: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpb4ro6vaj/q2iqtree.log
Date and Time: Fri Feb 28 11:27:41 2020
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-00veqgb9/c67b6543-8e61-435d-b61e-36a863284e6e/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpb4ro6vaj/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-scxhh8t5/c67b6543-8e61-435d-b61e-36a863284e6e/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpaz5m_db_/q2iqtree -nt 1
Seed: 340352 (Using SPRNG - Scalable Parallel Random Number Generator)
Time: Fri Feb 28 11:27:45 2020
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-scxhh8t5/c67b6543-8e61-435d-b61e-36a863284e6e/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, 1.228 / LogL: -1394.470
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.008, 1.302 / LogL: -1394.729
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.008, 1.305 / LogL: -1394.727
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.008, 1.301 / LogL: -1394.728
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.008, 1.305 / LogL: -1394.760
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.306 / LogL: -1394.786
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.008, 1.303 / LogL: -1394.730
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.304 / LogL: -1394.743
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.008, 1.305 / LogL: -1394.754
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.008, 1.311 / LogL: -1394.758
Optimal pinv,alpha: 0.000, 1.228 / LogL: -1394.470
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: -1392.888
--------------------------------------------------------------------
| INITIALIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.088 second
Computing log-likelihood of 98 initial trees ... 0.198 seconds
Current best score: -1392.888
Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.267
Iteration 10 / LogL: -1387.384 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.280 / Time: 0h:0m:1s
Finish initializing candidate tree set (2)
Current best tree score: -1387.267 / CPU time: 0.653
Number of iterations: 20
--------------------------------------------------------------------
| OPTIMIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Iteration 30 / LogL: -1387.348 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 40 / LogL: -1387.342 / Time: 0h:0m:1s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.258
Iteration 50 / LogL: -1387.346 / Time: 0h:0m:1s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.258
Iteration 60 / LogL: -1387.258 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 70 / LogL: -1387.386 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 80 / LogL: -1387.377 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 90 / LogL: -1387.347 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 100 / LogL: -1387.347 / 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: -1387.258
Optimal log-likelihood: -1387.254
Rate parameters: A-C: 0.32866 A-G: 2.26506 A-T: 2.14271 C-G: 1.17415 C-T: 3.29496 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: 1.315
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1387.254
Total tree length: 6.769
Total number of iterations: 102
CPU time used for tree search: 3.050 sec (0h:0m:3s)
Wall-clock time used for tree search: 3.062 sec (0h:0m:3s)
Total CPU time used: 3.554 sec (0h:0m:3s)
Total wall-clock time used: 3.569 sec (0h:0m:3s)
Analysis results written to:
IQ-TREE report: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpaz5m_db_/q2iqtree.iqtree
Maximum-likelihood tree: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpaz5m_db_/q2iqtree.treefile
Likelihood distances: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpaz5m_db_/q2iqtree.mldist
Screen log file: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpaz5m_db_/q2iqtree.log
Date and Time: Fri Feb 28 11:27:48 2020
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-scxhh8t5/c67b6543-8e61-435d-b61e-36a863284e6e/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpaz5m_db_/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-s_1xjkve/c67b6543-8e61-435d-b61e-36a863284e6e/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpjtozjjyf/q2iqtree -nt 1 -fast
Seed: 47350 (Using SPRNG - Scalable Parallel Random Number Generator)
Time: Fri Feb 28 11:27:53 2020
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-s_1xjkve/c67b6543-8e61-435d-b61e-36a863284e6e/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: 47350)
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, 1.240 / LogL: -1394.551
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.019, 1.306 / LogL: -1395.300
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.023, 1.366 / LogL: -1395.504
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.023, 1.367 / LogL: -1395.485
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.026, 1.365 / LogL: -1395.669
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.028, 1.365 / LogL: -1395.797
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.023, 1.365 / LogL: -1395.543
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.024, 1.367 / LogL: -1395.601
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.025, 1.367 / LogL: -1395.650
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.026, 1.367 / LogL: -1395.697
Optimal pinv,alpha: 0.000, 1.240 / LogL: -1394.551
Parameters optimization took 0.322 sec
Computing ML distances based on estimated model parameters... 0.003 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1392.871
--------------------------------------------------------------------
| INITIALIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.282
UPDATE BEST LOG-LIKELIHOOD: -1387.274
Finish initializing candidate tree set (3)
Current best tree score: -1387.274 / CPU time: 0.051
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: -1387.274
Optimal log-likelihood: -1387.259
Rate parameters: A-C: 0.33569 A-G: 2.26227 A-T: 2.13669 C-G: 1.17589 C-T: 3.29103 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: 1.304
Parameters optimization took 1 rounds (0.005 sec)
BEST SCORE FOUND : -1387.259
Total tree length: 6.801
Total number of iterations: 2
CPU time used for tree search: 0.050 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.051 sec (0h:0m:0s)
Total CPU time used: 0.397 sec (0h:0m:0s)
Total wall-clock time used: 0.401 sec (0h:0m:0s)
---> START RUN NUMBER 2 (seed: 48350)
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: -1482.894
2. Current log-likelihood: -1402.077
3. Current log-likelihood: -1396.805
4. Current log-likelihood: -1395.377
5. Current log-likelihood: -1394.645
Optimal log-likelihood: -1394.074
Rate parameters: A-C: 0.27529 A-G: 2.39359 A-T: 2.12128 C-G: 1.20984 C-T: 3.30250 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 (0.040 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: -1388.215
BETTER TREE FOUND at iteration 2: -1388.181
Finish initializing candidate tree set (4)
Current best tree score: -1388.181 / CPU time: 0.064
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: 0.36871 A-G: 2.32155 A-T: 2.12849 C-G: 1.22859 C-T: 3.29561 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 (0.026 sec)
BEST SCORE FOUND : -1387.535
Total tree length: 7.507
Total number of iterations: 2
CPU time used for tree search: 0.063 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.064 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)
---> START RUN NUMBER 3 (seed: 49350)
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: 0.080
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 (0.023 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: 0.161 sec (0h:0m:0s)
Total wall-clock time used: 0.165 sec (0h:0m:0s)
---> START RUN NUMBER 4 (seed: 50350)
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: -1479.269
2. Current log-likelihood: -1404.566
3. Current log-likelihood: -1399.236
4. Current log-likelihood: -1397.830
5. Current log-likelihood: -1397.061
Optimal log-likelihood: -1396.470
Rate parameters: A-C: 0.24622 A-G: 2.08129 A-T: 1.99444 C-G: 1.06443 C-T: 2.85655 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 (0.038 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: 0.33227 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 (0.024 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: 0.056 sec (0h:0m:0s)
Total CPU time used: 0.136 sec (0h:0m:0s)
Total wall-clock time used: 0.139 sec (0h:0m:0s)
---> START RUN NUMBER 5 (seed: 51350)
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: -1481.520
2. Current log-likelihood: -1401.794
3. Current log-likelihood: -1396.546
4. Current log-likelihood: -1395.117
5. Current log-likelihood: -1394.384
Optimal log-likelihood: -1393.812
Rate parameters: A-C: 0.27489 A-G: 2.41323 A-T: 2.17672 C-G: 1.25266 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 (0.038 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: 0.048
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: 0.35581 A-G: 2.35552 A-T: 2.14265 C-G: 1.20478 C-T: 3.37542 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: 0.048 sec (0h:0m:0s)
Total CPU time used: 0.133 sec (0h:0m:0s)
Total wall-clock time used: 0.136 sec (0h:0m:0s)
---> START RUN NUMBER 6 (seed: 52350)
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: -1486.149
2. Current log-likelihood: -1402.098
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: 0.27529 A-G: 2.39363 A-T: 2.12129 C-G: 1.20989 C-T: 3.30258 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: 0.059
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: -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: 0.36871 A-G: 2.32156 A-T: 2.12850 C-G: 1.22859 C-T: 3.29563 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 (0.026 sec)
BEST SCORE FOUND : -1387.535
Total tree length: 7.507
Total number of iterations: 2
CPU time used for tree search: 0.058 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.059 sec (0h:0m:0s)
Total CPU time used: 0.140 sec (0h:0m:0s)
Total wall-clock time used: 0.143 sec (0h:0m:0s)
---> START RUN NUMBER 7 (seed: 53350)
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: -1482.954
2. Current log-likelihood: -1402.081
3. Current log-likelihood: -1396.805
4. Current log-likelihood: -1395.378
5. Current log-likelihood: -1394.645
Optimal log-likelihood: -1394.074
Rate parameters: A-C: 0.27529 A-G: 2.39359 A-T: 2.12128 C-G: 1.20985 C-T: 3.30251 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 (0.040 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: 0.36871 A-G: 2.32155 A-T: 2.12849 C-G: 1.22859 C-T: 3.29561 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 (0.024 sec)
BEST SCORE FOUND : -1387.535
Total tree length: 7.507
Total number of iterations: 2
CPU time used for tree search: 0.063 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.063 sec (0h:0m:0s)
Total CPU time used: 0.147 sec (0h:0m:0s)
Total wall-clock time used: 0.150 sec (0h:0m:0s)
---> START RUN NUMBER 8 (seed: 54350)
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: -1482.403
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: 0.056
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 (0.024 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: 0.056 sec (0h:0m:0s)
Total CPU time used: 0.141 sec (0h:0m:0s)
Total wall-clock time used: 0.144 sec (0h:0m:0s)
---> START RUN NUMBER 9 (seed: 55350)
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: -1482.404
2. Current log-likelihood: -1403.041
3. Current log-likelihood: -1398.273
4. Current log-likelihood: -1396.900
5. Current log-likelihood: -1396.172
Optimal log-likelihood: -1395.670
Rate parameters: A-C: 0.23522 A-G: 2.07026 A-T: 1.96673 C-G: 1.07739 C-T: 2.84705 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: 1.338
Parameters optimization took 5 rounds (0.037 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 (4)
Current best tree score: -1388.172 / CPU time: 0.042
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: -1387.952
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: 0.33567 A-G: 2.25588 A-T: 2.13013 C-G: 1.17112 C-T: 3.26236 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 (0.025 sec)
BEST SCORE FOUND : -1387.418
Total tree length: 6.706
Total number of iterations: 2
CPU time used for tree search: 0.042 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.042 sec (0h:0m:0s)
Total CPU time used: 0.123 sec (0h:0m:0s)
Total wall-clock time used: 0.126 sec (0h:0m:0s)
---> START RUN NUMBER 10 (seed: 56350)
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: -1481.468
2. Current log-likelihood: -1401.790
3. Current log-likelihood: -1396.546
4. Current log-likelihood: -1395.117
5. Current log-likelihood: -1394.384
Optimal log-likelihood: -1393.812
Rate parameters: A-C: 0.27488 A-G: 2.41325 A-T: 2.17675 C-G: 1.25265 C-T: 3.29501 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 (0.035 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: 0.049
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: 0.35581 A-G: 2.35550 A-T: 2.14264 C-G: 1.20478 C-T: 3.37540 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: 0.049 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.049 sec (0h:0m:0s)
Total CPU time used: 0.131 sec (0h:0m:0s)
Total wall-clock time used: 0.134 sec (0h:0m:0s)
---> SUMMARIZE RESULTS FROM 10 RUNS
Run 1 gave best log-likelihood: -1387.259
Total CPU time for 10 runs: 1.671 seconds.
Total wall-clock time for 10 runs: 1.709 seconds.
Analysis results written to:
IQ-TREE report: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpjtozjjyf/q2iqtree.iqtree
Maximum-likelihood tree: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpjtozjjyf/q2iqtree.treefile
Trees from independent runs: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpjtozjjyf/q2iqtree.runtrees
Likelihood distances: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpjtozjjyf/q2iqtree.mldist
Screen log file: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpjtozjjyf/q2iqtree.log
Date and Time: Fri Feb 28 11:27:54 2020
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-s_1xjkve/c67b6543-8e61-435d-b61e-36a863284e6e/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpjtozjjyf/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-g3kizhqg/c67b6543-8e61-435d-b61e-36a863284e6e/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfk73zti6/q2iqtree -nt 1 -alrt 1000 -abayes -lbp 1000
Seed: 933482 (Using SPRNG - Scalable Parallel Random Number Generator)
Time: Fri Feb 28 11:27:58 2020
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-g3kizhqg/c67b6543-8e61-435d-b61e-36a863284e6e/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, 1.386 / LogL: -1392.892
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.008, 1.381 / LogL: -1392.857
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.386 / LogL: -1392.886
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.008, 1.377 / LogL: -1392.831
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.379 / LogL: -1392.845
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.008, 1.381 / 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 0.485 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... 0.091 second
Computing log-likelihood of 98 initial trees ... 0.207 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: -1387.256
Iteration 10 / LogL: -1387.269 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.289 / Time: 0h:0m:1s
Finish initializing candidate tree set (2)
Current best tree score: -1387.256 / CPU time: 0.615
Number of iterations: 20
--------------------------------------------------------------------
| OPTIMIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Iteration 30 / LogL: -1387.374 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 40 / LogL: -1387.374 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 50 / LogL: -1387.375 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 60 / LogL: -1387.352 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 70 / LogL: -1387.731 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 80 / LogL: -1387.381 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 90 / LogL: -1388.542 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 100 / LogL: -1387.370 / 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: -1387.256
Optimal log-likelihood: -1387.253
Rate parameters: A-C: 0.33248 A-G: 2.27708 A-T: 2.15579 C-G: 1.18365 C-T: 3.30920 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: 1.323
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...
0.057 sec.
Total tree length: 6.714
Total number of iterations: 102
CPU time used for tree search: 2.986 sec (0h:0m:2s)
Wall-clock time used for tree search: 2.989 sec (0h:0m:2s)
Total CPU time used: 3.551 sec (0h:0m:3s)
Total wall-clock time used: 3.557 sec (0h:0m:3s)
Analysis results written to:
IQ-TREE report: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfk73zti6/q2iqtree.iqtree
Maximum-likelihood tree: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfk73zti6/q2iqtree.treefile
Likelihood distances: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfk73zti6/q2iqtree.mldist
Screen log file: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfk73zti6/q2iqtree.log
Date and Time: Fri Feb 28 11:28:02 2020
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-g3kizhqg/c67b6543-8e61-435d-b61e-36a863284e6e/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfk73zti6/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-fx13358_/c67b6543-8e61-435d-b61e-36a863284e6e/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpdqj32h9n/q2iqtree -nt 1 -nstop 200 -pers 0.200000
Seed: 660724 (Using SPRNG - Scalable Parallel Random Number Generator)
Time: Fri Feb 28 11:28:06 2020
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-fx13358_/c67b6543-8e61-435d-b61e-36a863284e6e/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: -1416.636
2. Current log-likelihood: -1395.960
3. Current log-likelihood: -1390.000
4. Current log-likelihood: -1389.337
5. Current log-likelihood: -1389.140
6. Current log-likelihood: -1389.022
Optimal log-likelihood: -1388.947
Rate parameters: A-C: 0.15999 A-G: 2.04528 A-T: 1.56960 C-G: 0.79365 C-T: 2.04528 G-T: 1.00000
Base frequencies: A: 0.250 C: 0.250 G: 0.250 T: 0.250
Site proportion and rates: (0.668,0.376) (0.332,2.254)
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: -1389.067
--------------------------------------------------------------------
| INITIALIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.088 second
Computing log-likelihood of 97 initial trees ... 0.136 seconds
Current best score: -1388.947
Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1382.381
Iteration 10 / LogL: -1382.387 / Time: 0h:0m:0s
Iteration 20 / LogL: -1382.389 / Time: 0h:0m:0s
Finish initializing candidate tree set (1)
Current best tree score: -1382.381 / CPU time: 0.451
Number of iterations: 20
--------------------------------------------------------------------
| OPTIMIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
BETTER TREE FOUND at iteration 21: -1382.380
Iteration 30 / LogL: -1382.922 / Time: 0h:0m:0s (0h:0m:4s left)
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 37: -1382.036
Iteration 40 / LogL: -1382.871 / Time: 0h:0m:0s (0h:0m:3s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.034
Iteration 50 / LogL: -1388.652 / Time: 0h:0m:0s (0h:0m:3s left)
Iteration 60 / LogL: -1382.045 / Time: 0h:0m:0s (0h:0m:2s left)
Iteration 70 / LogL: -1382.438 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 80 / LogL: -1382.437 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 90 / LogL: -1382.439 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 100 / LogL: -1382.438 / Time: 0h:0m:1s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.034
Iteration 110 / LogL: -1382.437 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 120 / LogL: -1382.437 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 130 / LogL: -1382.438 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 140 / LogL: -1382.105 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 150 / LogL: -1382.436 / Time: 0h:0m:1s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.034
Iteration 160 / LogL: -1382.104 / Time: 0h:0m:1s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.034
Iteration 170 / LogL: -1382.104 / Time: 0h:0m:2s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.034
Iteration 180 / LogL: -1382.185 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 190 / LogL: -1382.437 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 200 / LogL: -1382.225 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 210 / LogL: -1382.108 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 220 / LogL: -1382.106 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 230 / LogL: -1382.104 / Time: 0h:0m:2s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 238 ITERATIONS / Time: 0h:0m:2s
--------------------------------------------------------------------
| FINALIZING TREE SEARCH |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1382.034
Optimal log-likelihood: -1382.026
Rate parameters: A-C: 0.18964 A-G: 1.83455 A-T: 1.53204 C-G: 0.76868 C-T: 1.83455 G-T: 1.00000
Base frequencies: A: 0.250 C: 0.250 G: 0.250 T: 0.250
Site proportion and rates: (0.718,0.416) (0.282,2.491)
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1382.026
Total tree length: 6.982
Total number of iterations: 238
CPU time used for tree search: 2.705 sec (0h:0m:2s)
Wall-clock time used for tree search: 2.711 sec (0h:0m:2s)
Total CPU time used: 2.759 sec (0h:0m:2s)
Total wall-clock time used: 2.767 sec (0h:0m:2s)
Analysis results written to:
IQ-TREE report: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpdqj32h9n/q2iqtree.iqtree
Maximum-likelihood tree: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpdqj32h9n/q2iqtree.treefile
Likelihood distances: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpdqj32h9n/q2iqtree.mldist
Screen log file: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpdqj32h9n/q2iqtree.log
Date and Time: Fri Feb 28 11:28:10 2020
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-fx13358_/c67b6543-8e61-435d-b61e-36a863284e6e/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpdqj32h9n/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-zlvwycxz/c67b6543-8e61-435d-b61e-36a863284e6e/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpi9rhim1o/q2iqtreeufboot -nt 1 -nstop 200 -pers 0.200000
Seed: 580991 (Using SPRNG - Scalable Parallel Random Number Generator)
Time: Fri Feb 28 11:28:14 2020
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-zlvwycxz/c67b6543-8e61-435d-b61e-36a863284e6e/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: -1416.738
2. Current log-likelihood: -1396.507
3. Current log-likelihood: -1389.944
4. Current log-likelihood: -1389.313
5. Current log-likelihood: -1389.127
6. Current log-likelihood: -1389.021
Optimal log-likelihood: -1388.956
Rate parameters: A-C: 0.16337 A-G: 2.05004 A-T: 1.55714 C-G: 0.78643 C-T: 2.05004 G-T: 1.00000
Base frequencies: A: 0.250 C: 0.250 G: 0.250 T: 0.250
Site proportion and rates: (0.668,0.373) (0.332,2.263)
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: -1389.065
--------------------------------------------------------------------
| INITIALIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.087 second
Computing log-likelihood of 98 initial trees ... 0.133 seconds
Current best score: -1388.851
Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1382.373
UPDATE BEST LOG-LIKELIHOOD: -1382.372
Iteration 10 / LogL: -1382.373 / Time: 0h:0m:0s
Iteration 20 / LogL: -1382.381 / Time: 0h:0m:0s
Finish initializing candidate tree set (1)
Current best tree score: -1382.372 / CPU time: 0.618
Number of iterations: 20
--------------------------------------------------------------------
| OPTIMIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
Estimate model parameters (epsilon = 0.100)
UPDATE BEST LOG-LIKELIHOOD: -1382.104
Iteration 30 / LogL: -1382.113 / Time: 0h:0m:0s (0h:0m:4s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.103
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 34: -1382.024
Iteration 40 / LogL: -1382.434 / Time: 0h:0m:1s (0h:0m:5s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.023
Iteration 50 / LogL: -1382.040 / Time: 0h:0m:1s (0h:0m:4s left)
Log-likelihood cutoff on original alignment: -1400.814
Iteration 60 / LogL: -1382.437 / Time: 0h:0m:1s (0h:0m:4s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.022
Iteration 70 / LogL: -1382.025 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 80 / LogL: -1382.434 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 90 / LogL: -1382.118 / Time: 0h:0m:2s (0h:0m:3s left)
Iteration 100 / LogL: -1382.758 / Time: 0h:0m:2s (0h:0m:3s left)
Log-likelihood cutoff on original alignment: -1399.570
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.997
Iteration 110 / LogL: -1382.105 / Time: 0h:0m:2s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.022
Iteration 120 / LogL: -1382.097 / Time: 0h:0m:2s (0h:0m:2s left)
Iteration 130 / LogL: -1382.434 / Time: 0h:0m:2s (0h:0m:2s left)
Iteration 140 / LogL: -1382.024 / Time: 0h:0m:3s (0h:0m:2s left)
Iteration 150 / LogL: -1382.059 / Time: 0h:0m:3s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1401.179
Iteration 160 / LogL: -1382.097 / Time: 0h:0m:3s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.022
Iteration 170 / LogL: -1382.025 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 180 / LogL: -1382.147 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 190 / LogL: -1382.309 / Time: 0h:0m:4s (0h:0m:0s left)
Iteration 200 / LogL: -1382.032 / Time: 0h:0m:4s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1402.144
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.998
UPDATE BEST LOG-LIKELIHOOD: -1382.022
Iteration 210 / LogL: -1382.455 / Time: 0h:0m:4s (0h:0m:1s left)
Iteration 220 / LogL: -1382.062 / Time: 0h:0m:4s (0h:0m:1s left)
Iteration 230 / LogL: -1382.033 / Time: 0h:0m:4s (0h:0m:1s left)
TREE SEARCH COMPLETED AFTER 235 ITERATIONS / Time: 0h:0m:5s
--------------------------------------------------------------------
| FINALIZING TREE SEARCH |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1382.022
Optimal log-likelihood: -1382.016
Rate parameters: A-C: 0.19114 A-G: 1.83092 A-T: 1.52678 C-G: 0.76544 C-T: 1.83092 G-T: 1.00000
Base frequencies: A: 0.250 C: 0.250 G: 0.250 T: 0.250
Site proportion and rates: (0.720,0.414) (0.280,2.505)
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1382.016
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpi9rhim1o/q2iqtreeufboot.splits.nex
Total tree length: 7.011
Total number of iterations: 235
CPU time used for tree search: 4.922 sec (0h:0m:4s)
Wall-clock time used for tree search: 4.954 sec (0h:0m:4s)
Total CPU time used: 5.055 sec (0h:0m:5s)
Total wall-clock time used: 5.093 sec (0h:0m:5s)
Computing bootstrap consensus tree...
Reading input file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpi9rhim1o/q2iqtreeufboot.splits.nex...
20 taxa and 145 splits.
Consensus tree written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpi9rhim1o/q2iqtreeufboot.contree
Reading input trees file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpi9rhim1o/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1382.016
Analysis results written to:
IQ-TREE report: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpi9rhim1o/q2iqtreeufboot.iqtree
Maximum-likelihood tree: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpi9rhim1o/q2iqtreeufboot.treefile
Likelihood distances: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpi9rhim1o/q2iqtreeufboot.mldist
Ultrafast bootstrap approximation results written to:
Split support values: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpi9rhim1o/q2iqtreeufboot.splits.nex
Consensus tree: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpi9rhim1o/q2iqtreeufboot.contree
Screen log file: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpi9rhim1o/q2iqtreeufboot.log
Date and Time: Fri Feb 28 11:28:20 2020
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-zlvwycxz/c67b6543-8e61-435d-b61e-36a863284e6e/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpi9rhim1o/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-jzcm2io3/c67b6543-8e61-435d-b61e-36a863284e6e/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpsv18dn9z/q2iqtreeufboot -nt 1 -alrt 1000 -abayes -lbp 1000 -nstop 200 -pers 0.200000
Seed: 818587 (Using SPRNG - Scalable Parallel Random Number Generator)
Time: Fri Feb 28 11:28:24 2020
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-jzcm2io3/c67b6543-8e61-435d-b61e-36a863284e6e/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: 818587)...
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, 1.281 / 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: 0.008, 1.382 / LogL: -1392.826
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.009, 1.386 / LogL: -1392.893
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, 1.378 / LogL: -1392.845
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.008, 1.383 / LogL: -1392.851
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 0.478 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... 0.084 second
Computing log-likelihood of 98 initial trees ... 0.210 seconds
Current best score: -1389.881
Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.263
Iteration 10 / LogL: -1387.284 / Time: 0h:0m:1s
Iteration 20 / LogL: -1387.275 / Time: 0h:0m:1s
Finish initializing candidate tree set (1)
Current best tree score: -1387.263 / CPU time: 0.749
Number of iterations: 20
--------------------------------------------------------------------
| OPTIMIZING CANDIDATE TREE SET |
--------------------------------------------------------------------
BETTER TREE FOUND at iteration 27: -1387.260
BETTER TREE FOUND at iteration 29: -1387.260
Iteration 30 / LogL: -1387.266 / Time: 0h:0m:1s (0h:0m:10s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.260
Iteration 40 / LogL: -1387.345 / Time: 0h:0m:1s (0h:0m:8s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.259
Iteration 50 / LogL: -1387.563 / Time: 0h:0m:2s (0h:0m:7s left)
Log-likelihood cutoff on original alignment: -1406.007
Iteration 60 / LogL: -1387.283 / Time: 0h:0m:2s (0h:0m:6s left)
Iteration 70 / LogL: -1387.360 / Time: 0h:0m:2s (0h:0m:6s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.259
Iteration 80 / LogL: -1387.259 / Time: 0h:0m:2s (0h:0m:5s left)
Iteration 90 / LogL: -1387.333 / Time: 0h:0m:3s (0h:0m:4s left)
Iteration 100 / LogL: -1387.359 / Time: 0h:0m:3s (0h:0m:4s left)
Log-likelihood cutoff on original alignment: -1407.608
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.995
Iteration 110 / LogL: -1387.299 / Time: 0h:0m:3s (0h:0m:4s left)
Iteration 120 / LogL: -1387.359 / Time: 0h:0m:4s (0h:0m:3s left)
Iteration 130 / LogL: -1387.307 / Time: 0h:0m:4s (0h:0m:3s left)
Iteration 140 / LogL: -1387.360 / Time: 0h:0m:4s (0h:0m:3s left)
Iteration 150 / LogL: -1387.352 / Time: 0h:0m:5s (0h:0m:2s left)
Log-likelihood cutoff on original alignment: -1408.422
Iteration 160 / LogL: -1387.370 / Time: 0h:0m:5s (0h:0m:2s left)
Iteration 170 / LogL: -1387.527 / Time: 0h:0m:5s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.259
Iteration 180 / LogL: -1387.278 / Time: 0h:0m:5s (0h:0m:1s left)
Iteration 190 / LogL: -1387.363 / Time: 0h:0m:6s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.259
Iteration 200 / LogL: -1387.527 / Time: 0h:0m:6s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1408.422
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.999
Iteration 210 / LogL: -1387.324 / Time: 0h:0m:6s (0h:0m:2s left)
Iteration 220 / LogL: -1387.361 / Time: 0h:0m:7s (0h:0m:2s left)
Iteration 230 / LogL: -1388.510 / Time: 0h:0m:7s (0h:0m:2s left)
TREE SEARCH COMPLETED AFTER 230 ITERATIONS / Time: 0h:0m:7s
--------------------------------------------------------------------
| FINALIZING TREE SEARCH |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.259
Optimal log-likelihood: -1387.254
Rate parameters: A-C: 0.33716 A-G: 2.26442 A-T: 2.13773 C-G: 1.17713 C-T: 3.28833 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: 1.312
Parameters optimization took 1 rounds (0.006 sec)
BEST SCORE FOUND : -1387.254
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...
0.061 sec.
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpsv18dn9z/q2iqtreeufboot.splits.nex
Total tree length: 6.763
Total number of iterations: 230
CPU time used for tree search: 7.017 sec (0h:0m:7s)
Wall-clock time used for tree search: 7.043 sec (0h:0m:7s)
Total CPU time used: 7.661 sec (0h:0m:7s)
Total wall-clock time used: 7.692 sec (0h:0m:7s)
Computing bootstrap consensus tree...
Reading input file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpsv18dn9z/q2iqtreeufboot.splits.nex...
20 taxa and 151 splits.
Consensus tree written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpsv18dn9z/q2iqtreeufboot.contree
Reading input trees file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpsv18dn9z/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1387.254
Analysis results written to:
IQ-TREE report: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpsv18dn9z/q2iqtreeufboot.iqtree
Maximum-likelihood tree: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpsv18dn9z/q2iqtreeufboot.treefile
Likelihood distances: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpsv18dn9z/q2iqtreeufboot.mldist
Ultrafast bootstrap approximation results written to:
Split support values: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpsv18dn9z/q2iqtreeufboot.splits.nex
Consensus tree: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpsv18dn9z/q2iqtreeufboot.contree
Screen log file: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpsv18dn9z/q2iqtreeufboot.log
Date and Time: Fri Feb 28 11:28:32 2020
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-jzcm2io3/c67b6543-8e61-435d-b61e-36a863284e6e/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpsv18dn9z/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!