Great, thanks! I’ll give this a bash!
I think I have this working! Quick question: which output file should I use for downstream humann3 analysis? I think it’s either seq_kneaddata.fastq (reads that were not in the reference database) or seq_kneaddata.trimmed.fastq (trimmed reads) - probably the first one?
Many thanks!
Hello, Glad to hear you think you have it working! Yes, use the first file you listed. That should be the file that has the reads which have run through all of the quality control steps. If you want to double check to confirm look at the end of the kneaddata log for that run and it should list the final output file at the end. The “trimmed” file that you listed are just those reads that have passed through the trimmomatic step.
Thank you,
Lauren
Perfect, thank you so much
Hello again! kneaddata has worked and I’ve just started running humann3 on the kneaddata.fastq files, but it’s coming up with the following:
(biobakery3) rantwis@uos-p-bioi-02:~/seqdata/clean_danish for f in *.fastq; do humann -i $f -o outputs; done
Output files will be written to: /home/rantwis/seqdata/clean_danish/outputs
WARNING: Can not call software version for bowtie2
Running metaphlan …
Total species selected from prescreen: 0
Selected species explain 0.00% of predicted community composition
No species were selected from the prescreen.
Because of this the custom ChocoPhlAn database is empty.
This will result in zero species-specific gene families and pathways.
Running diamond …
Aligning to reference database: uniref90_201901.dmnd
Would kneaddata have replaced an older version of bowtie perhaps? Or is it a problem with the kneaddata files?
Many thanks
Hello - Thanks for the follow up and glad to hear that kneaddata worked with your custom tree database! You usually do not need to worry about the warning for the bowtie2 version call. Some bowtie2 conda installs have a slightly different version output then expected by HUMAnN. If your kneaddata runs did not result in any errors (and it sounds like they were okay) then the output files should be okay. You might just have gotten a slightly different bowtie2 version when you installed kneaddata. If you want to double check bowtie2, run “$ bowtie2 --version” and make sure that you are running a version newer than v2.1.
Thank you,
Lauren
Thanks Lauren - I’ve had a look at the output files, the genefamilies.tsv file looks ok, e.g. head:
| # Gene Family | Ash100.unmapped_kneaddata_Abundance-RPKs |
|---|---|
| UNMAPPED | 3909204 |
| UniRef90_UPI000C1CF1DF | 13511.16763 |
| UniRef90_UPI000C1CF1DF | unclassified |
| UniRef90_UPI000C1CD3EA | 10899.74546 |
| UniRef90_UPI000C1CD3EA | unclassified |
| UniRef90_Q3LVI5 | 10505.13876 |
| UniRef90_Q3LVI5 | unclassified |
| UniRef90_UPI000C1D281F | 7110.48856 |
but the pathcoverage.tsv shows:
| # Pathway | Ash100.unmapped_kneaddata_Coverage |
|---|---|
| UNMAPPED | 1 |
| UNINTEGRATED | 1 |
and the pathabundance.tsv shows:
| # Pathway | Ash100.unmapped_kneaddata_Abundance |
|---|---|
| UNMAPPED | 0 |
| UNINTEGRATED | 0 |
Does this seem right to you?
Many thanks
Hi - Thanks for the detailed post! It might be okay as long as you did not see any errors in your run. It is likely that there were just not enough reactions for any of the pathways at significant coverage to call any pathways.
Thank you,
Lauren
Thanks Lauren, I don’t think there are any errors (log file pasted below) - the only problem I think is that from the data produced I won’t be able to tell which microbes are in there, and which one is doing what - I’ll only have the gene pathways, which isn’t quite what we wanted. Do you know if there’s a work around for this? Many thanks.
Log file:
02:~/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp$ less Ash10.unmapped_kneaddata.log
09/30/2020 11:21:47 PM - humann.humann - INFO: Running humann v3.0.0.alpha.3
09/30/2020 11:21:47 PM - humann.humann - INFO: Output files will be written to: /home/rantwis/seqdata/clean_danish/outputs
09/30/2020 11:21:47 PM - humann.humann - INFO: Writing temp files to directory: /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp
09/30/2020 11:21:47 PM - humann.utilities - INFO: File ( /home/rantwis/seqdata/clean_danish/Ash10.unmapped_kneaddata.fastq ) is of format: fastq
09/30/2020 11:21:47 PM - humann.utilities - DEBUG: Check software, metaphlan, for required version, 3.0
09/30/2020 11:21:47 PM - humann.utilities - INFO: Using metaphlan version 3.0
09/30/2020 11:21:47 PM - humann.utilities - DEBUG: Check software, bowtie2, for required version, 2.2
09/30/2020 11:21:48 PM - humann.utilities - WARNING: Can not call software version for bowtie2
09/30/2020 11:21:48 PM - humann.utilities - INFO: Using bowtie2 version UNK
09/30/2020 11:21:48 PM - humann.humann - INFO: Search mode set to uniref90 because a uniref90 translated search database is selected
09/30/2020 11:21:48 PM - humann.utilities - DEBUG: Check software, diamond, for required version, 0.9.24
09/30/2020 11:21:48 PM - humann.utilities - INFO: Using diamond version 2.0.4
09/30/2020 11:21:48 PM - humann.config - INFO:
Run config settings:
DATABASE SETTINGS
nucleotide database folder = /home/rantwis/miniconda3/envs/biobakery3/bin/chocophlan
protein database folder = /home/rantwis/miniconda3/envs/biobakery3/bin/uniref
pathways database file 1 = /home/rantwis/miniconda3/envs/biobakery3/lib/python3.7/site-packages/humann/data/pathways/metacyc_reactions_level4ec_only.uniref.bz2
pathways database file 2 = /home/rantwis/miniconda3/envs/biobakery3/lib/python3.7/site-packages/humann/data/pathways/metacyc_pathways_structured_filtered
utility mapping database folder = /home/rantwis/miniconda3/envs/biobakery3/bin/utility_mapping
RUN MODES
resume = False
verbose = False
bypass prescreen = False
bypass nucleotide index = False
bypass nucleotide search = False
bypass translated search = False
translated search = diamond
pick frames = off
threads = 1
SEARCH MODE
search mode = uniref90
nucleotide identity threshold = 0.0
translated identity threshold = 80.0
ALIGNMENT SETTINGS
bowtie2 options = --very-sensitive
diamond options = --top 1 --outfmt 6
evalue threshold = 1.0
prescreen threshold = 0.01
translated subject coverage threshold = 50.0
translated query coverage threshold = 90.0
nucleotide subject coverage threshold = 50.0
nucleotide query coverage threshold = 90.0
PATHWAYS SETTINGS
minpath = on
xipe = off
gap fill = on
INPUT AND OUTPUT FORMATS
input file format = fastq
output file format = tsv
output max decimals = 10
remove stratified output = False
remove column description output = False
log level = DEBUG
09/30/2020 11:21:48 PM - humann.store - DEBUG: Initialize Alignments class instance to minimize memory use
09/30/2020 11:21:48 PM - humann.store - DEBUG: Initialize Reads class instance to minimize memory use
09/30/2020 11:22:05 PM - humann.humann - INFO: Load pathways database part 1: /home/rantwis/miniconda3/envs/biobakery3/lib/python3.7/site-packages/humann/data/pathways/metacyc_reactions_level4ec_only.uniref.bz2
09/30/2020 11:22:06 PM - humann.humann - INFO: Load pathways database part 2: /home/rantwis/miniconda3/envs/biobakery3/lib/python3.7/site-packages/humann/data/pathways/metacyc_pathways_structured_filtered
09/30/2020 11:22:06 PM - humann.search.prescreen - INFO: Running metaphlan …
09/30/2020 11:22:06 PM - humann.utilities - DEBUG: Using software: /home/rantwis/miniconda3/envs/biobakery3/bin/metaphlan
09/30/2020 11:22:06 PM - humann.utilities - DEBUG: Remove file: /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/Ash10.unmapped_kneaddata_metaphlan_bugs_list.tsv
09/30/2020 11:22:06 PM - humann.utilities - DEBUG: Remove file: /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/Ash10.unmapped_kneaddata_metaphlan_bowtie2.txt
09/30/2020 11:22:06 PM - humann.utilities - INFO: Execute command: /home/rantwis/miniconda3/envs/biobakery3/bin/metaphlan /home/rantwis/seqdata/clean_danish/Ash10.unmapped_kneaddata.fastq -t rel_ab -o /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/Ash10.unmapped_kneaddata_metaphlan_bugs_list.tsv --input_type fastq --bowtie2out /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/Ash10.unmapped_kneaddata_metaphlan_bowtie2.txt
09/30/2020 11:24:40 PM - humann.utilities - DEBUG: b’Use of uninitialized value $bt2_args[2] in join or string at /home/rantwis/miniconda3/envs/biobakery3/bin/bowtie2 line 423.\nUse of uninitialized value bt2_args[3] in join or string at /home/rantwis/miniconda3/envs/biobakery3/bin/bowtie2 line 423.\nUse of uninitialized value [2] in string eq at /home/rantwis/miniconda3/envs/biobakery3/bin/bowtie2 line 360.\nUse of uninitialized value $[3] in string eq at /home/rantwis/miniconda3/envs/biobakery3/bin/bowtie2 line 360.\nUse of uninitialized value in exists at /home/rantwis/miniconda3/envs/biobakery3/bin/bowtie2 line 81.\nUse of uninitialized value in exists at /home/rantwis/miniconda3/envs/biobakery3/bin/bowtie2 line 81.\nUse of uninitialized value $bt2_args[2] in join or string at /home/rantwis/miniconda3/envs/biobakery3/bin/bowtie2 line 459.\nUse of uninitialized value $bt2_args[3] in join or string at /home/rantwis/miniconda3/envs/biobakery3/bin/bowtie2 line 459.\n’
09/30/2020 11:24:40 PM - humann.humann - INFO: TIMESTAMP: Completed prescreen : 155 seconds
09/30/2020 11:24:40 PM - humann.search.prescreen - INFO: Total species selected from prescreen: 0
09/30/2020 11:24:40 PM - humann.search.prescreen - DEBUG:
No species were selected from the prescreen.
Because of this the custom ChocoPhlAn database is empty.
This will result in zero species-specific gene families and pathways.
09/30/2020 11:24:40 PM - humann.humann - INFO: TIMESTAMP: Completed custom database creation : 0 seconds
09/30/2020 11:24:40 PM - humann.humann - DEBUG: Custom database is empty
09/30/2020 11:24:40 PM - humann.store - DEBUG: Initialize Reads class instance to minimize memory use
09/30/2020 11:25:35 PM - humann.utilities - DEBUG: Remove file: /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/tmp7r2imfsd/tmpr08ymww0
09/30/2020 11:25:35 PM - humann.search.translated - DEBUG: Convert unaligned reads fastq file to fasta
09/30/2020 11:26:17 PM - humann.utilities - DEBUG: Remove file: /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/Ash10.unmapped_kneaddata_diamond_aligned.tsv
09/30/2020 11:26:17 PM - humann.search.translated - INFO: Running diamond …
09/30/2020 11:26:17 PM - humann.search.translated - INFO: Aligning to reference database: uniref90_201901.dmnd
09/30/2020 11:26:17 PM - humann.utilities - DEBUG: Remove file: /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/tmp7r2imfsd/diamond_m8_uiwz072b
09/30/2020 11:26:17 PM - humann.utilities - DEBUG: Using software: /home/rantwis/miniconda3/envs/biobakery3/bin/diamond
09/30/2020 11:26:17 PM - humann.utilities - INFO: Execute command: /home/rantwis/miniconda3/envs/biobakery3/bin/diamond blastx --query /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/tmp7r2imfsd/tmp0b5j_3ue --evalue 1.0 --threads 1 --top 1 --outfmt 6 --db /home/rantwis/miniconda3/envs/biobakery3/bin/uniref/uniref90_201901 --out /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/tmp7r2imfsd/diamond_m8_uiwz072b --tmpdir /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/tmp7r2imfsd
10/01/2020 03:44:44 AM - humann.utilities - DEBUG: b’diamond v2.0.4.142 © Max Planck Society for the Advancement of Science\nDocumentation, support and updates available at http://www.diamondsearch.org\n\n#CPU threads: 1\nScoring parameters: (Matrix=BLOSUM62 Lambda=0.267 K=0.041 Penalties=11/1)\nTemporary directory: /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/tmp7r2imfsd\nOpening the database… [0.092s]\nPercentage range of top alignment score to report hits: 1\nReference = /home/rantwis/miniconda3/envs/biobakery3/bin/uniref/uniref90_201901.dmnd\nSequences = 87296736\nLetters = 29247941583\nBlock size = 2000000000\nOpening the input file… [0.04s]\nOpening the output file… [0s]\nLoading query sequences… [7.063s]\nMasking queries… [53.163s]\nBuilding query seed set… [0.092s]\n\x1b[1;33mThe host system is detected to have 540 GB of RAM. It is recommended to increase the block size for better performance using these parameters : -b12 -c1\n\x1b[0;39mAlgorithm: Double-indexed\nBuilding query histograms… [7.375s]\nAllocating buffers… [0s]\nLoading reference sequences… [4.125s]\nMasking reference… [113.528s]\nInitializing temporary storage… [0s]\nBuilding reference histograms… [43.987s]\nAllocating buffers… [0s]\nProcessing query block 1, reference block 1/15, shape 1/2, index chunk 1/4.\nBuilding reference seed array… [32.455s]\nBuilding query seed array… [5.49s]\nComputing hash join… [14.909s]\nBuilding seed filter… [0.549s]\nSearching alignments… [19.914s]\nProcessing query block 1, reference block 1/15, shape 1/2, index chunk 2/4.\nBuilding reference seed array… [40.37s]\nBuilding query seed array… [6.411s]\nComputing hash join… [13.861s]\nBuilding seed filter… [0.558s]\nSearching alignments… [19.714s]\nProcessing query block 1, reference block 1/15, shape 1/2, index chunk 3/4.\nBuilding reference seed array… [42.066s]\nBuilding query seed array… [6.737s]\nComputing hash join… [13.863s]\nBuilding seed filter… [0.547s]\nSearching alignments… [20.707s]\nProcessing query block 1, reference block 1/15, shape 1/2, index chunk 4/4.\nBuilding reference seed array… [31.675s]\nBuilding query seed array… [5.516s]\nComputing hash join… [13.631s]\nBuilding seed filter… [0.545s]\nSearching alignments… [22.354s]\nProcessing query block 1, reference block 1/15, shape 2/2, index chunk 1/4.\nBuilding reference seed array… [31.401s]\nBuilding query seed array… [4.924s]\nComputing hash join… [13.052s]\nBuilding seed filter… [0.483s]\nSearching alignments… [18.284s]\nProcessing query block 1, reference block 1/15, shape 2/2, index chunk 2/4.\nBuilding reference seed array… [40.415s]\nBuilding query seed array… [5.768s]\nComputing hash join… [13.209s]\nBuilding seed filter… [0.494s]\nSearching alignments… [17.395s]\nProcessing query block 1, reference block 1/15, shape 2/2, index chunk 3/4.\nBuilding reference seed array… [42.033s]\nBuilding query seed array… [5.801s]\nComputing hash join… [12.546s]\nBuilding seed filter… [0.463s]\nSearching alignments… [17.001s]\nProcessing query block 1, reference block 1/15, shape 2/2, index chunk 4/4.\nBuilding reference seed array… [31.145s]\nBuilding query seed array… [4.801s]\nComputing hash join… [12.773s]\nBuilding seed filter… [0.446s]\nSearching alignments… [17.068s]\nDeallocating buffers…Searching alignments… [12.996s]\nProcessing query block 1, reference block 15/15, shape 2/2, index chunk 3/4.\nBuilding reference seed array… [30.64s]\nBuilding query seed array… [6.86s]\nComputing hash join… [8.6s]\nBuilding seed filter… [0.442s]\nSearching alignments… [12.884s]\nProcessing query block 1, reference block 15/15, shape 2/2, index chunk 4/4.\nBuilding reference seed array… [22.692s]\nBuilding query seed array… [5.636s]\nComputing hash join… [8.765s]\nBuilding seed filter… [0.447s]\nSearching alignments… [13.494s]\nDeallocating buffers… [0.245s]\nClearing query masking… [0.376s]\nOpening temporary output file… [0s]\nComputing alignments… [144.214s]\nDeallocating reference… [0.108s]\nLoading reference sequences… [0s]\nDeallocating buffers… [0.032s]\nJoining output blocks… [12.807s]\nDeallocating queries… [0.304s]\nLoading query sequences… [0s]\nClosing the input file… [0.004s]\nClosing the output file… [0.047s]\nClosing the database file… [0.004s]\nDeallocating taxonomy… [0s]\nTotal time = 15506.8s\nReported 4251950 pairwise alignments, 4251950 HSPs.\n869762 queries aligned.\n\x1b[1;33mThe host system is detected to have 540 GB of RAM. It is recommended to increase the block size for better performance using these parameters : -b12 -c1\n\x1b[0;39m’
10/01/2020 03:44:44 AM - humann.utilities - DEBUG: Using software: /bin/cat
10/01/2020 03:44:44 AM - humann.utilities - INFO: Execute command: /bin/cat /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/tmp7r2imfsd/diamond_m8_uiwz072b
10/01/2020 03:44:45 AM - humann.utilities - DEBUG: Remove file: /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/tmp7r2imfsd/tmp0b5j_3ue
10/01/2020 03:44:45 AM - humann.humann - INFO: TIMESTAMP: Completed translated alignment : 15604 seconds
10/01/2020 03:45:37 AM - humann.utilities - DEBUG: Total alignments where percent identity is not a number: 0
10/01/2020 03:45:37 AM - humann.utilities - DEBUG: Total alignments where alignment length is not a number: 0
10/01/2020 03:45:37 AM - humann.utilities - DEBUG: Total alignments where E-value is not a number: 0
10/01/2020 03:45:37 AM - humann.utilities - DEBUG: Total alignments not included based on large e-value: 0
10/01/2020 03:45:37 AM - humann.utilities - DEBUG: Total alignments not included based on small percent identity: 370291
10/01/2020 03:45:37 AM - humann.utilities - DEBUG: Total alignments not included based on small query coverage: 1369055
10/01/2020 03:45:37 AM - humann.search.blastx_coverage - INFO: Total alignments without coverage information: 0
10/01/2020 03:45:37 AM - humann.search.blastx_coverage - INFO: Total proteins in blastx output: 156361
10/01/2020 03:45:37 AM - humann.search.blastx_coverage - INFO: Total proteins without lengths: 0
10/01/2020 03:45:37 AM - humann.search.blastx_coverage - INFO: Proteins with coverage greater than threshold (50.0): 5446
10/01/2020 03:46:30 AM - humann.utilities - DEBUG: Total alignments where percent identity is not a number: 0
10/01/2020 03:46:30 AM - humann.utilities - DEBUG: Total alignments where alignment length is not a number: 0
10/01/2020 03:46:30 AM - humann.utilities - DEBUG: Total alignments where E-value is not a number: 0
10/01/2020 03:46:30 AM - humann.utilities - DEBUG: Total alignments not included based on large e-value: 0
10/01/2020 03:46:30 AM - humann.utilities - DEBUG: Total alignments not included based on small percent identity: 370291
10/01/2020 03:46:30 AM - humann.utilities - DEBUG: Total alignments not included based on small query coverage: 1369055
10/01/2020 03:46:30 AM - humann.search.translated - DEBUG: Total translated alignments not included based on small subject coverage value: 1838236
10/01/2020 03:47:37 AM - humann.utilities - DEBUG: Remove file: /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/tmp7r2imfsd/tmpn6irn3xj
10/01/2020 03:47:39 AM - humann.humann - INFO: TIMESTAMP: Completed translated alignment post-processing : 175 seconds
10/01/2020 03:47:39 AM - humann.humann - INFO: Total bugs after translated alignment: 1
10/01/2020 03:47:40 AM - humann.humann - INFO:
unclassified: 799546 hits
10/01/2020 03:47:40 AM - humann.humann - INFO: Total gene families after translated alignment: 5446
10/01/2020 03:47:40 AM - humann.humann - INFO: Unaligned reads after translated alignment: 96.1618525301 %
10/01/2020 03:47:40 AM - humann.humann - INFO: Computing gene families …
10/01/2020 03:47:40 AM - humann.quantify.families - DEBUG: Compute gene families
10/01/2020 03:47:42 AM - humann.store - INFO:
Total gene families : 5446
unclassified : 5446 gene families
10/01/2020 03:47:58 AM - humann.humann - INFO: TIMESTAMP: Completed computing gene families : 18 seconds
10/01/2020 03:47:58 AM - humann.humann - INFO: Computing pathways abundance and coverage …
10/01/2020 03:47:58 AM - humann.quantify.modules - DEBUG: Write flat reactions to pathways file for Minpath
10/01/2020 03:47:58 AM - humann.quantify.modules - INFO: Compute reaction scores for bug: unclassified
10/01/2020 03:48:01 AM - humann.quantify.modules - INFO: Run MinPath on unclassified
10/01/2020 03:48:01 AM - humann.quantify.modules - INFO: Compute reaction scores for bug: all
10/01/2020 03:48:03 AM - humann.quantify.modules - INFO: Run MinPath on all
10/01/2020 03:48:03 AM - humann.utilities - DEBUG: Using python module : /home/rantwis/miniconda3/envs/biobakery3/lib/python3.7/site-packages/humann/quantify/MinPath12hmp.py
10/01/2020 03:48:03 AM - humann.utilities - INFO: Execute command: /home/rantwis/miniconda3/envs/biobakery3/bin/python /home/rantwis/miniconda3/envs/biobakery3/lib/python3.7/site-packages/humann/quantify/MinPath12hmp.py -any /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/tmp7r2imfsd/tmpd9u69cmz -map /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/tmp7r2imfsd/tmp8ijgoryi -report /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/tmp7r2imfsd/tmpvewov1ea -details /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/tmp7r2imfsd/tmptk0e7gdk -mps /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/tmp7r2imfsd/tmp8i2k7gyc
10/01/2020 03:48:03 AM - humann.utilities - DEBUG: Using python module : /home/rantwis/miniconda3/envs/biobakery3/lib/python3.7/site-packages/humann/quantify/MinPath12hmp.py
10/01/2020 03:48:03 AM - humann.utilities - INFO: Execute command: /home/rantwis/miniconda3/envs/biobakery3/bin/python /home/rantwis/miniconda3/envs/biobakery3/lib/python3.7/site-packages/humann/quantify/MinPath12hmp.py -any /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/tmp7r2imfsd/tmpek57gr7h -map /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/tmp7r2imfsd/tmp8ijgoryi -report /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/tmp7r2imfsd/tmpwfl29gg8 -details /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/tmp7r2imfsd/tmp1xdkonfu -mps /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/tmp7r2imfsd/tmp_7s3am_1
10/01/2020 03:48:04 AM - humann.quantify.modules - DEBUG: Compute pathway abundance for bug: unclassified
10/01/2020 03:48:04 AM - humann.quantify.modules - DEBUG: Compute pathway abundance for bug: all
10/01/2020 03:48:04 AM - humann.quantify.modules - DEBUG: Compute pathway coverage for bug: unclassified
10/01/2020 03:48:04 AM - humann.quantify.modules - DEBUG: Compute pathway coverage for bug: all
10/01/2020 03:48:04 AM - humann.quantify.modules - DEBUG: Print pathways _Abundance
10/01/2020 03:48:04 AM - humann.quantify.modules - DEBUG: Print pathways _Coverage
10/01/2020 03:48:04 AM - humann.humann - INFO: TIMESTAMP: Completed computing pathways : 6 seconds
10/01/2020 03:48:04 AM - humann.humann - INFO:
Output files created:
/home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_genefamilies.tsv
/home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_pathabundance.tsv
/home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_pathcoverage.tsv
10/01/2020 03:48:04 AM - humann.utilities - DEBUG: Remove directory: /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/tmp7r2imfsd
Hello, Thank you for posting the log. HUMAnN calls MetaPhlAn to determine the predicted community composition. It looks like from the log there might possibly have been an error in running MetaPhlAn (or from its call of bowtie2) and so no species were identified for the run. Would you try running MetaPhlAn directly (with the command below) and check for any unexpected errors? If it is an error from bowtie2 I would suggest reinstalling bowtie2 and rerunning the MetaPhlAn command just to make sure the bowtie2 error is resolved. If so, then try rerunning HUMAnN.
$ /home/rantwis/miniconda3/envs/biobakery3/bin/metaphlan /home/rantwis/seqdata/clean_danish/Ash10.unmapped_kneaddata.fastq -t rel_ab -o /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/Ash10.unmapped_kneaddata_metaphlan_bugs_list.tsv --input_type fastq --bowtie2out /home/rantwis/seqdata/clean_danish/outputs/Ash10.unmapped_kneaddata_humann_temp/Ash10.unmapped_kneaddata_metaphlan_bowtie2.txt
Thank you,
Lauren
Many thanks! It’s saying:
OSError: fatal error running ‘/home/rantwis/miniconda3/envs/biobakery3/lib/python3.7/site-packages/metaphlan/utils/read_fastx.py’. Is it in the system path?
It is in the system path, but this doesn’t seem like a bowtie error?
Hi - It looks like possibly an error with your MetaPhlAn install. If read_fastx.py is in the system path can you try running the executable with the command $ read_fastx.py and see if it returns an error message? I wonder if maybe there is a dependency needed for that script that might not be installed.
Thank you,
Lauren
Thanks, it’s saying:
read_fastx.py: command not found
What’s weird is this was all working fine before I installed kneaddata, so perhaps there is something about kneaddata that is confusing humann3? I have reinstalled humann3 to see if that fixed it but it didn’t - perhaps I should delete everything including kneaddata and rebuild humann3?
Thanks
Hi - That executable is only needed for MetaPhlAn. It is possible that some of your installs might have changed when you installed kneaddata. My suggestion would be to uninstall and then reinstall MetaPhlAn. Then try running MetaPhlAn again and hopefully this should have fixed it.
Thank you,
Lauren
Alas, no such luck. I’ll remove kneaddata and humann3 and then reinstall humann3 and hopefully that will work. Many thanks.
Hello - If the reinstall does not work, check your metaphlan install version. I wonder if you might have a slightly older version that gets installed by default in your conda environment. You might need to specify that the latest version be installed so you do not get the conda default.
Thank you,
Lauren
Ok thanks, yeah it’s still not working. Is 3.0.2 the most up to date version of MetaPhlAn?
Thanks
Hi - I just checked and the latest metaphlan version is v3.0.4. Try upgrading to the latest version to see if it resolves the error you are seeing.
Thank you,
Lauren
Ok thanks - I can’t seem to update it past v3.0.2 using either
conda install MetaPhlAn==3.0.4 or
conda install -c bioconda python=3.7 metaphlan
The various installs and deletions seem to have also confused humann3 so I’m deleting and reinstalling again. If you have any suggestions on how to force the 3.0.4 install they would be very welcome!
Thanks
P.S. The install says it is installing metaphlan bioconda/noarch::metaphlan-3.0.4-pyh7b7c402_0
but after the install, --version indicates it is version 3.0.2