Error: duplicate keys

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Melanie Smee

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May 10, 2017, 10:17:30 AM5/10/17
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Hi there,

I'm pretty new to these analyses and using Linux in general, so please forgive my naivety! I've installed Phylophlan and its dependencies. When using the example dataset, I end up with an empty treefile but I just then used the alignment file generated with another tree-building program (IQTree). That's a problem for another day! Otherwise it ran fine.

Now that I'm using my own data (15 bacterial genomes downloaded from NCBI) I'm coming across the following error:

Traceback (most recent call last):

  File "./phylophlan.py", line 803, in <module>

    aln_merge(projn,pars['integrate'])

  File "./phylophlan.py", line 505, in aln_merge

    faas = SeqIO.to_dict( faas )

  File "/Users/Shared/biopython-1.68/Bio/SeqIO/__init__.py", line 734, in to_dict

    raise ValueError("Duplicate key '%s'" % key)

ValueError: Duplicate key 'WP_003339565.1'


Using grep I found that there's quite a few duplicates!

dhcp-morris-3090:phylophlan mrs353$ grep  ">" ./data/syringae_genomes/*.sub.aln |  awk '{print $1}' | sort | uniq -c | awk '{if($1>1) print}'

   2 ./data/syringae_genomes/p0007.sub.aln:>WP_003339565.1

   2 ./data/syringae_genomes/p0008.sub.aln:>WP_003317946.1

   2 ./data/syringae_genomes/p0010.sub.aln:>WP_003317110.1

   2 ./data/syringae_genomes/p0013.sub.aln:>WP_003364819.1

   2 ./data/syringae_genomes/p0017.sub.aln:>WP_003340175.1

   2 ./data/syringae_genomes/p0021.sub.aln:>WP_003345654.1

   2 ./data/syringae_genomes/p0026.sub.aln:>WP_002555138.1

   2 ./data/syringae_genomes/p0026.sub.aln:>WP_003395180.1

   2 ./data/syringae_genomes/p0029.sub.aln:>WP_003314194.1

   2 ./data/syringae_genomes/p0030.sub.aln:>WP_003367943.1

   2 ./data/syringae_genomes/p0031.sub.aln:>WP_003345368.1

   2 ./data/syringae_genomes/p0031.sub.aln:>WP_003363893.1

   3 ./data/syringae_genomes/p0033.sub.aln:>WP_003316986.1

   2 ./data/syringae_genomes/p0034.sub.aln:>WP_003372639.1

   2 ./data/syringae_genomes/p0036.sub.aln:>WP_003340565.1

   2 ./data/syringae_genomes/p0037.sub.aln:>WP_002555022.1

   2 ./data/syringae_genomes/p0038.sub.aln:>WP_003317104.1

   2 ./data/syringae_genomes/p0044.sub.aln:>WP_002554688.1

   2 ./data/syringae_genomes/p0048.sub.aln:>WP_002554661.1

   2 ./data/syringae_genomes/p0049.sub.aln:>WP_002551973.1

   2 ./data/syringae_genomes/p0051.sub.aln:>WP_003366319.1

   2 ./data/syringae_genomes/p0052.sub.aln:>WP_002555041.1

   2 ./data/syringae_genomes/p0056.sub.aln:>WP_003394970.1

   3 ./data/syringae_genomes/p0059.sub.aln:>WP_003367019.1

   2 ./data/syringae_genomes/p0060.sub.aln:>WP_003317209.1

   2 ./data/syringae_genomes/p0061.sub.aln:>WP_002555469.1

   2 ./data/syringae_genomes/p0062.sub.aln:>WP_002555499.1

   3 ./data/syringae_genomes/p0065.sub.aln:>WP_003314293.1

   2 ./data/syringae_genomes/p0074.sub.aln:>WP_003316710.1

   2 ./data/syringae_genomes/p0076.sub.aln:>WP_003368786.1

   2 ./data/syringae_genomes/p0078.sub.aln:>WP_002552735.1

   2 ./data/syringae_genomes/p0081.sub.aln:>WP_002555464.1

   2 ./data/syringae_genomes/p0083.sub.aln:>WP_002552734.1

   2 ./data/syringae_genomes/p0084.sub.aln:>WP_003317125.1

   2 ./data/syringae_genomes/p0085.sub.aln:>WP_002555078.1

   2 ./data/syringae_genomes/p0086.sub.aln:>WP_002555984.1

   2 ./data/syringae_genomes/p0086.sub.aln:>WP_003315957.1

   2 ./data/syringae_genomes/p0087.sub.aln:>WP_003347797.1

   2 ./data/syringae_genomes/p0088.sub.aln:>WP_002555982.1

   2 ./data/syringae_genomes/p0095.sub.aln:>WP_003318486.1

   2 ./data/syringae_genomes/p0097.sub.aln:>WP_003390862.1

   2 ./data/syringae_genomes/p0099.sub.aln:>WP_002555477.1

   2 ./data/syringae_genomes/p0103.sub.aln:>WP_003340582.1

   2 ./data/syringae_genomes/p0107.sub.aln:>WP_002551458.1

   2 ./data/syringae_genomes/p0108.sub.aln:>WP_002551820.1

   2 ./data/syringae_genomes/p0110.sub.aln:>WP_003339591.1

   2 ./data/syringae_genomes/p0117.sub.aln:>WP_003368841.1

   2 ./data/syringae_genomes/p0119.sub.aln:>WP_002555235.1

   2 ./data/syringae_genomes/p0124.sub.aln:>WP_002551560.1

   2 ./data/syringae_genomes/p0132.sub.aln:>WP_003391089.1

   2 ./data/syringae_genomes/p0135.sub.aln:>WP_003318510.1

   2 ./data/syringae_genomes/p0140.sub.aln:>WP_002555991.1

   2 ./data/syringae_genomes/p0143.sub.aln:>WP_003365095.1

   2 ./data/syringae_genomes/p0145.sub.aln:>WP_002555108.1

   2 ./data/syringae_genomes/p0152.sub.aln:>WP_003343817.1

   2 ./data/syringae_genomes/p0154.sub.aln:>WP_003363593.1

   2 ./data/syringae_genomes/p0154.sub.aln:>WP_003412906.1

   2 ./data/syringae_genomes/p0157.sub.aln:>WP_003317384.1

   2 ./data/syringae_genomes/p0162.sub.aln:>WP_003363707.1

   2 ./data/syringae_genomes/p0170.sub.aln:>WP_003342804.1

   2 ./data/syringae_genomes/p0176.sub.aln:>WP_002551999.1

   3 ./data/syringae_genomes/p0178.sub.aln:>WP_002552523.1

   2 ./data/syringae_genomes/p0179.sub.aln:>WP_003340692.1

   2 ./data/syringae_genomes/p0180.sub.aln:>WP_003314296.1

   2 ./data/syringae_genomes/p0181.sub.aln:>WP_003365366.1

   2 ./data/syringae_genomes/p0183.sub.aln:>WP_002555744.1

   3 ./data/syringae_genomes/p0187.sub.aln:>WP_003316562.1

   2 ./data/syringae_genomes/p0194.sub.aln:>WP_003339485.1

   3 ./data/syringae_genomes/p0198.sub.aln:>WP_003316561.1

   2 ./data/syringae_genomes/p0201.sub.aln:>WP_002554013.1

   2 ./data/syringae_genomes/p0202.sub.aln:>WP_003390978.1

   2 ./data/syringae_genomes/p0205.sub.aln:>WP_002555327.1

   2 ./data/syringae_genomes/p0208.sub.aln:>WP_003396212.1

   2 ./data/syringae_genomes/p0209.sub.aln:>WP_003304938.1

   2 ./data/syringae_genomes/p0213.sub.aln:>WP_003339743.1

   2 ./data/syringae_genomes/p0218.sub.aln:>WP_003313606.1

   2 ./data/syringae_genomes/p0218.sub.aln:>WP_003364105.1

   2 ./data/syringae_genomes/p0219.sub.aln:>WP_003344468.1

   3 ./data/syringae_genomes/p0230.sub.aln:>WP_003364825.1

   2 ./data/syringae_genomes/p0233.sub.aln:>WP_003369901.1

   2 ./data/syringae_genomes/p0253.sub.aln:>WP_003318491.1

   2 ./data/syringae_genomes/p0255.sub.aln:>WP_003364457.1

   2 ./data/syringae_genomes/p0262.sub.aln:>WP_002554022.1

   2 ./data/syringae_genomes/p0266.sub.aln:>WP_003396266.1

   2 ./data/syringae_genomes/p0268.sub.aln:>WP_003316734.1

   2 ./data/syringae_genomes/p0268.sub.aln:>WP_003366142.1

   2 ./data/syringae_genomes/p0270.sub.aln:>WP_002555089.1

   2 ./data/syringae_genomes/p0270.sub.aln:>WP_003313594.1

   2 ./data/syringae_genomes/p0271.sub.aln:>WP_002551508.1

   2 ./data/syringae_genomes/p0272.sub.aln:>WP_003368689.1

   2 ./data/syringae_genomes/p0273.sub.aln:>WP_003371726.1

   2 ./data/syringae_genomes/p0280.sub.aln:>WP_003363717.1

   2 ./data/syringae_genomes/p0283.sub.aln:>WP_003313989.1

   2 ./data/syringae_genomes/p0285.sub.aln:>WP_003316635.1

   2 ./data/syringae_genomes/p0286.sub.aln:>WP_003365583.1

   3 ./data/syringae_genomes/p0294.sub.aln:>WP_002551326.1

   3 ./data/syringae_genomes/p0297.sub.aln:>WP_003318426.1

   2 ./data/syringae_genomes/p0304.sub.aln:>WP_003364017.1

   2 ./data/syringae_genomes/p0307.sub.aln:>WP_003393371.1

   2 ./data/syringae_genomes/p0309.sub.aln:>WP_003316641.1

   2 ./data/syringae_genomes/p0318.sub.aln:>WP_003415201.1

   2 ./data/syringae_genomes/p0323.sub.aln:>WP_003394873.1

   2 ./data/syringae_genomes/p0326.sub.aln:>WP_003316351.1

   2 ./data/syringae_genomes/p0332.sub.aln:>WP_003392250.1

   2 ./data/syringae_genomes/p0344.sub.aln:>WP_003313553.1

   2 ./data/syringae_genomes/p0348.sub.aln:>WP_003367855.1

   2 ./data/syringae_genomes/p0363.sub.aln:>WP_003366703.1

   2 ./data/syringae_genomes/p0364.sub.aln:>WP_003314434.1

   2 ./data/syringae_genomes/p0366.sub.aln:>WP_003314231.1

   2 ./data/syringae_genomes/p0369.sub.aln:>WP_003340639.1

   2 ./data/syringae_genomes/p0370.sub.aln:>WP_002555093.1

   3 ./data/syringae_genomes/p0374.sub.aln:>WP_003316803.1

   2 ./data/syringae_genomes/p0377.sub.aln:>WP_003367573.1

   2 ./data/syringae_genomes/p0378.sub.aln:>WP_003317954.1

   3 ./data/syringae_genomes/p0379.sub.aln:>WP_003346110.1

   2 ./data/syringae_genomes/p0380.sub.aln:>WP_003314085.1

   2 ./data/syringae_genomes/p0383.sub.aln:>WP_003393775.1

   2 ./data/syringae_genomes/p0386.sub.aln:>WP_003393629.1


I'm not really sure where to go from here - if the problem originates further back in the process how do I deal with it? I've tried cleaning the temporary files and re-doing it and get the same each time. I also get the same problem when I run it on our University server with their versions of the program and its dependencies.

Many thanks for any help at all!
Mel




Francesco Asnicar

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May 10, 2017, 5:24:47 PM5/10/17
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Hello Mel,
first of all, many thanks for using PhyloPhlAn.

Let's start with the last error you reported. The "duplicate key" error it is because of the inputs. You did a search inside the data folder and there is ok if you find more than one occurrence in different files. I think that if you search for the same key in your input folder, you'll find at least two different genomes using the same id, and that's the problem that makes PhyloPhlAn stop!
One thing that I can suggest you is to try the PhyloPhlAn in the dev branch of the repository. From the "phylophlan" folder (the one you cloned form the repository) you can type "hg up dev". You can use the same command you used in the original version, but here there is an extra function that is taking care of cleaning the input ids. So you'll just need to add the "--faa_cleaning" param to your old command and run PhyloPhlAn again.
With the dev version of PhyloPhlAn you can use a newer version of usearch, and I recommend to use the latest available.

About the first problem you reported, which input example you used? Can you provide the command you used?

Please let me know if you have other problems.

Many thanks,
Francesco

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Melanie Smee

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May 11, 2017, 11:31:53 AM5/11/17
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Hi Francesco,

Many thanks for your reply - that seems to have done the trick! I'm now getting a similar error to what I got with the example dataset, so I think perhaps there's a problem with FastTree... The command I used was ./phylophlan.py -u syringae_genomes --faa_cleaning

Building the phylogenetic tree with FastTree

Traceback (most recent call last):

  File "./phylophlan.py", line 1719, in <module>

    build_phylo_tree(projn, pars['integrate'], pars['nproc'], pars['raxml'])

  File "./phylophlan.py", line 1130, in build_phylo_tree

    t.wait()

AttributeError: 'int' object has no attribute 'wait'


Thanks again for all your help!
Mel

Francesco Asnicar

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May 11, 2017, 12:08:51 PM5/11/17
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Hi Mel,
I updated the code of PhyloPhlAn (in the dev branch) that should fix your problem. To update your local version, it should be enough to type "hg up" from the "phylophlan" repository folder.
Thanks for pointing out this issue.

Please, let me know if there are any other problems.

Thanks,
Francesco

Melanie Smee

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May 11, 2017, 2:30:04 PM5/11/17
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Hi Francesco,

Thanks again for your quick response! I downloaded phylophlan again since you did the updates and re-ran the analysis. Unfortunately the newick treefile is still empty but I got no warning:

All alignments merged into data/syringae_genomes/aln.fna!

output/syringae_genomes/aln_stats.tsv generated!

Building the phylogenetic tree with FastTree

Tree built! The output newick file is in output/syringae_genomes/

Tree building finished in 0 secs (2280 total time).


Sorry! I'm not sure why this is still happening - any thoughts much appreciated.
Mel

Francesco Asnicar

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May 11, 2017, 3:08:38 PM5/11/17
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Hi Mel,
I think that FastTree is not working the way it should. Can you try execute this command:
FastTree -fastest -mlnni 4 -spr 4 -mlacc 2 -slownni -no2nd -out output/syringae_genomes/syringae_genomes.nwk data/syringae_genomes/aln.fna

If you get an error (as I'm expecting), this is the reason why PhyloPhlAn does not produce a tree. If you can report the error, then I can try to fix this in the PhyloPhlAn code.

Many thanks,
Francesco

Melanie Smee

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May 11, 2017, 3:56:03 PM5/11/17
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Hi Francesco, 

OK, the error message I get from that code is:

FastTree Version 2.1.9 SSE3

Alignment: data/syringae_genomes/aln.fna

Amino acid distances: BLOSUM45 Joins: balanced Support: SH-like 1000

Search: Fastest +NNI +SPR (4 rounds range 10) +ML-NNI (4 rounds) opt-each=2

TopHits: 1.00*sqrtN close=default refresh=0.50

ML Model: Jones-Taylor-Thorton, CAT approximation with 20 rate categories

Segmentation fault: 11


Thanks again!

Mel


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Francesco Asnicar

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May 11, 2017, 5:09:35 PM5/11/17
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Hi Mel,
ok, so there is something wrong with FastTree. I tried with a couple of example, but I was not able to get the segmentation fault error from FastTree on my computer. Can you send me your alignment (data/syringae_genomes/aln.fna) so I can try to see if there is something wrong with that file?

Many thanks,
Francesco

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Melanie Smee

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May 12, 2017, 11:28:00 AM5/12/17
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Hi Francesco, 

Sure - thanks very much for all your help on this. File is attached. 

Best wishes, 
Mel

Francesco

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aln.fna

Francesco Asnicar

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May 12, 2017, 12:04:05 PM5/12/17
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Hi Mel,
thank you very much for the alignment file. I ran this command:
FastTree -fastest -mlnni 4 -spr 4 -mlacc 2 -slownni -no2nd -out aln.nwk aln.fna

and this is the output I got:
FastTree Version 2.1.9 SSE3
Alignment: aln.fna
Amino acid distances: BLOSUM45 Joins: balanced Support: SH-like 1000
Search: Fastest +NNI +SPR (4 rounds range 10) +ML-NNI (4 rounds) opt-each=2
TopHits: 1.00*sqrtN close=default refresh=0.50
ML Model: Jones-Taylor-Thorton, CAT approximation with 20 rate categories
Initial topology in 0.05 seconds
Refining topology: 16 rounds ME-NNIs, 4 rounds ME-SPRs, 4 rounds ML-NNIs
Total branch-length 2.432 after 1.04 sec6, 1 of 13 splits 
ML-NNI round 1: LogLk = -46286.809 NNIs 2 max delta 70.97 Time 2.39
Switched to using 20 rate categories (CAT approximation)18 of 20 
Rate categories were divided by 0.825 so that average rate = 1.0
CAT-based log-likelihoods may not be comparable across runs
Use -gamma for approximate but comparable Gamma(20) log-likelihoods
ML-NNI round 2: LogLk = -44971.262 NNIs 0 max delta 0.00 Time 3.93
Turning off heuristics for final round of ML NNIs (converged)
ML-NNI round 3: LogLk = -44971.258 NNIs 0 max delta 0.00 Time 5.22 (final)
Optimize all lengths: LogLk = -44971.258 Time 5.52
Total time: 6.44 seconds Unique: 15/15 Bad splits: 0/12

as well as the tree file (attached). I wasn't sure if it was due to the alignment file or FastTree, but now I think that your FastTree installation is not correct. Can you check you FastTree installation?

Many thanks,
Francesco

Francesco

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aln.nwk

Melanie Smee

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May 15, 2017, 9:26:51 AM5/15/17
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Hi Francesco, 

Thanks for your help on this. I've tried downloading/installing FastTree again, and when I run this command gcc -O3 -finline-functions -funroll-loops -Wall -o FastTree FastTree.c -lm

I get this warning (which I also got with the previous FastTree installation): 

clang: warning: optimization flag '-finline-functions' is not supported

If I just type in FastTree to check the installation I get the following:

Usage for FastTree version 2.1.10 SSE3:
  FastTree protein_alignment > tree
  FastTree < protein_alignment > tree
  FastTree -out tree protein_alignment
  FastTree -nt nucleotide_alignment > tree
  FastTree -nt -gtr < nucleotide_alignment > tree
  FastTree < nucleotide_alignment > tree
FastTree accepts alignments in fasta or phylip interleaved formats

Common options (must be before the alignment file):
  -quiet to suppress reporting information
  -nopr to suppress progress indicator
  -log logfile -- save intermediate trees, settings, and model details
  -fastest -- speed up the neighbor joining phase & reduce memory usage
        (recommended for >50,000 sequences)
  -n <number> to analyze multiple alignments (phylip format only)
        (use for global bootstrap, with seqboot and CompareToBootstrap.pl)
  -nosupport to not compute support values
  -intree newick_file to set the starting tree(s)
  -intree1 newick_file to use this starting tree for all the alignments
        (for faster global bootstrap on huge alignments)
  -pseudo to use pseudocounts (recommended for highly gapped sequences)
  -gtr -- generalized time-reversible model (nucleotide alignments only)
  -lg -- Le-Gascuel 2008 model (amino acid alignments only)
  -wag -- Whelan-And-Goldman 2001 model (amino acid alignments only)
  -quote -- allow spaces and other restricted characters (but not ' ) in
           sequence names and quote names in the output tree (fasta input only;
           FastTree will not be able to read these trees back in)
  -noml to turn off maximum-likelihood
  -nome to turn off minimum-evolution NNIs and SPRs
        (recommended if running additional ML NNIs with -intree)
  -nome -mllen with -intree to optimize branch lengths for a fixed topology
  -cat # to specify the number of rate categories of sites (default 20)
      or -nocat to use constant rates
  -gamma -- after optimizing the tree under the CAT approximation,
      rescale the lengths to optimize the Gamma20 likelihood
  -constraints constraintAlignment to constrain the topology search
       constraintAlignment should have 1s or 0s to indicates splits
  -expert -- see more options

I am still getting the segmentation fault:11 warning as well when I try to use FastTree with the alignment file. Any thoughts much appreciated!

Thanks, 
Mel

Francesco

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Francesco Asnicar

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May 15, 2017, 2:28:28 PM5/15/17
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Hi Mel,
the command you provided is the one for compiling FastTree for Mac and I have never used FastTree on a Mac, I always used it on a Linux computer.
I usually download both "Linux 64-bit executable (+SSE)" and "Multi-threaded executable (+SSE +OpenMP)", and depending on the size of the alignment file I decide which one to use.
Have you tried to compile it using the other command they suggest to use?
gcc -DNO_SSE -O3 -finline-functions -funroll-loops -Wall -o FastTree FastTree.c -lm

Many thanks,
Francesco

Francesco

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