During anvi-profile Config Error: Read.trim leads to indefinite hanging

Gsmith535 commented 4 years ago

Dear Anvio devs,

The Anvio version on our server was updated to 6.2 on 20-05-29, and re-installed today as a possible fix for the issue we are experiencing. As far as I know, Anvio was installed according to the conda installation directions (but I can get back to you on that after the weekend). Our cluster is running Ubuntu 18.04.4 LTS (Bionic Beaver) and the workload is managed by SLURM.

$ anvi-self-test --version
Anvi'o version ...............................: esther (v6.2)
Profile DB version ...........................: 31
Contigs DB version ...........................: 14
Pan DB version ...............................: 13
Genome data storage version ..................: 6
Auxiliary data storage version ...............: 2
Structure DB version .........................: 1

Since this update, multiple users on our cluster have encountered a new error when running anvi-profile.

Traceback (most recent call last):
  File "/usr/local/bioinfo/Anaconda3/envs/anvio6/lib/python3.6/multiprocessing/process.py", line 258, in _bootstrap
    self.run()
  File "/usr/local/bioinfo/Anaconda3/envs/anvio6/lib/python3.6/multiprocessing/process.py", line 93, in run
    self._target(*self._args, **self._kwargs)
  File "/usr/local/bioinfo/Anaconda3/envs/anvio6/lib/python3.6/site-packages/anvio/profiler.py", line 595, in profile_contig_worker
    contig = self.process_contig(bam_file, contig_name, contig_length)
  File "/usr/local/bioinfo/Anaconda3/envs/anvio6/lib/python3.6/site-packages/anvio/profiler.py", line 659, in process_contig
    split.auxiliary.process(bam_file)
  File "/usr/local/bioinfo/Anaconda3/envs/anvio6/lib/python3.6/site-packages/anvio/contigops.py", line 191, in process
    self.run_SNVs(bam)
  File "/usr/local/bioinfo/Anaconda3/envs/anvio6/lib/python3.6/site-packages/anvio/contigops.py", line 414, in run_SNVs
    for read in bam.fetch_and_trim(self.split.parent, self.split.start, self.split.end):
  File "/usr/local/bioinfo/Anaconda3/envs/anvio6/lib/python3.6/site-packages/anvio/bamops.py", line 83, in fetch_and_trim
    read.trim(trim_by=(read.reference_end - end), side='right')
  File "/usr/local/bioinfo/Anaconda3/envs/anvio6/lib/python3.6/site-packages/anvio/bamops.py", line 269, in trim
    " range of %d" % (trim_by, self.reference_end - self.reference_start))
anvio.errors.ConfigError: 

Config Error: Read.trim :: Requesting to trim an amount 818 that exceeds the alignment range
              of 251

After the error presents itself, the profiling continues until the second to last contig, and then hangs indefinitely (>23 hours when similar sized samples ran in minutes). Ctrl-C killing of the profiling leaves a clear scar in the Profile/RUNLOG.txt, "[DATE and TIME] * Anvi'o profiler received SIGINT, terminating all processes...", which is not present in the logs of normally completed profiles but otherwise appears normal. Although The error seems to be due to read length (full error below main text), but the recruited read lengths do not appear different between mapped reads that were able to profile and those that fail and do not match the requested amount or alignment range (e.g. 350 bp reads for this sample, see link for data below), and the requested amounts and alignment range appeared variable.

The error seems to occur somewhat stochastically - for multiple sets of reads mapped to contigs co-assembled from the same reads, only some mapped reads fail to generate a profile while others work fine. Furthermore, the same reads consistently fail to profile to the same contigs after re-mapping. This has been occurring for multiple different users working on completely independent projects, and some of these users have been working with anvio for months without this issue. As far as I know, nobody in our group has tried to use these profiles, for example merging or interactive sessions.

I noticed in the news that there was mention of a critical update for anvio 6.2 as of yesterday, 2020-06-04 (https://github.com/merenlab/anvio/blob/master/NEWS.md). If this issue should be resolved by updating using the instructions under "Following the active codebase (you’re a wizard, arry)" on the install site, please let me know and close the issue. I couldn't find any additional information about this specific issue or what was fixed in the critical update, and a fresh re-install did not resolve the error.

Flat text files for relevant data (mapping stats) and outputs (stderr) are available in this repo: https://github.com/Gsmith535/AnvioError_200605 I can make the contigs and reads available if necessary, but I would rather not due to their sizes.

Thank you for the wonderful software and excellent support! -G

meren commented 4 years ago

Dear @Gsmith535,

Apologies for this as our new profiler is slowly reaching to its stability. If I remember correctly, @ekiefl addressed this issue, but it is not available in a stable release yet. But I might be wrong, so testing this using the master branch is essential.

It would be great if you were to follow these instructions and let us know if the problem persists:

http://merenlab.org/2016/06/26/installation-v2/#following-the-active-codebase-youre-a-wizard-arry

Best,

ekiefl commented 4 years ago

Looking forward to hearing if this is persistent on the master branch. If not, please upload a bam file and contigs db that cause the error.

Gsmith535 commented 4 years ago

Hi @meren and @ekiefl , Thank you for the rapid responses. Our cluster is part of a larger conglomeration of other clusters, thus we are experiencing administrative difficulties in making the “master” environment as you suggested, but we are still trying. We have freshly reinstalled v6.2 and made sure to use the preferred methods on the installation page (conda install -y -c bioconda -c conda-forge anvio-minimal==6.2=py_1), but the error remains. We have re-installed v6.1 and no error occurs, consistent with the same data profiling properly in the past before the update to v6.2. Could you link me to the previous issue mentioned please? I have been unable to find anything regarding this error. I have uploaded the contigs, contigs database, and sorted/indexed bam files to an FTP site: https://ftp.science.ru.nl/MicrobiolNijmegen/AnvioIssue1436_ContBam.tar I have added descriptions of the files on the FTP site to REAMDE in the repo: https://github.com/Gsmith535/AnvioError_200605 Please let me know if the data are not available or anything else that could help resolve the issue.

ekiefl commented 4 years ago

Thank you @Gsmith535 for the comprehensive ftp server. I cannot find the past issue, but I will try and solve this problem today if it is not fixed in master.

If you cannot install master, perhaps we can consider a small release. @meren what do you think? If this is an unaddressed bug (not fixed in master), I think it means there are more profile bugs waiting to be reported and we should wait. But otherwise people will keep getting unfairly stuck here because they don't have master.

meren commented 4 years ago

Good point, @ekiefl. I just checked and found out that there are more than 900 commits between master and v6.2. I think it is a good idea to make a minor release that would support a stable 6 branch until we are ready for v7. I will try to develop a roadmap next week and discuss with everyone :)

ekiefl commented 4 years ago

OK. I have identified the problem. Almost all of the reads in this bam file look like this:

<anvio.bamops.Read object at 0x14e1699e8>
 ├── start, end : [169093, 169348)
 ├── cigartuple : [(7, 255)]
 ├── read       : CACAAGGAGGCGCGCATGAAAGTCGTTTCGATCCTGCTGGCACTGATCAACTTCCTCGCGGGGGTTTTACTTATCCTCTCCTGCATCTCGTCGAACGAAACATTCGCCTGGGTCGCATGGAAAACCGCTTTGGGATTCATGGGCGTGGCGTTCGGCATCCTGACCTTCAAAGACAGCGCCCAGCCCGTCGCTCAATGCAGGATGATTCTCTACGGCCTATTGCTCGTCATCGCCGGTGTCTCCATCGTCGCCTAT
 └── reference  : NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

The reason that the reference sequence is composed of Ns is because the mapping strategy did not include 'MD' tags. That means the reference sequence is not stored, so anvi'o just puts Ns. That's fine, but I just wanted to point that out because otherwise it makes it seem like something is wrong.

Here is the read that fails:

<anvio.bamops.Read object at 0x14e2336a0>
 ├── start, end : [166346, 190510)
 ├── cigartuple : [(7, 4), (8, 3), (7, 1), (1, 11), (7, 5), (8, 1), (7, 1), (8, 1), (7, 2), (8, 1), (7, 8), (2, 2), (7, 5), (2, 23879), (7, 251)]
 ├── read       : CCAATAAAGGTCTGTTTGGATGATTCTTTGCCACTTCA--GCAAC-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------GGCGCGGATCTGGTCGTCCACTCGATGACAAAATATCTCGGCGGTCACTCGGATGTGGTCGGCGGCGCGATCATTGGCAAAGACAAGGAACTTGGGGAAAAACTCGCCTATCTGCAAAACGCAATCGGCGGCGTGCTGGGACCGATGGACTCGTTCCTCGTGCTGCGCGGAATCAAAACATTACCCGTCCGCATGGATCGTCACGCCGAGAATGCTGAAAAAATTGTCGCATTTTTGGAGAAGCAGAAAAA
 └── reference  : NNNNNNNN-----------NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN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Wow. Whether it is garbage or interesting, I will leave to the scientists. Regardless, anvi'o should in theory be able to handle such a bizarre mapping...

Working on a fix now.

meren commented 4 years ago

cigartuple : [(7, 4), (8, 3), (7, 1), (1, 11), (7, 5), (8, 1), (7, 1), (8, 1), (7, 2), (8, 1), (7, 8), (2, 2), (7, 5), (2, 23879), (7, 251)]

Ouch. This looks extremely unusual for a mapping software to report for a short read. I am sorry if I missing something obvious, but can you remind us how did you generate these BAM files, @Gsmith535?

ekiefl commented 4 years ago

Anvi'o crashes because the portion of the read spanning the split in question is 20,580 consecutive - characters. An extremely fringe case. Never did I expect to be safeguarding against something like this lol.

Gsmith535 commented 4 years ago

Those are some odd looking read mappings, and they look like they should have been removed by either QC/trimming/filtering or by failure to align. We updated our version to v6.2 on 2020-05-27, after which multiple users noticed this issue and never before. I had used this exact same combination of contigs+reads successfully on 2020-05-26, immediately before our update, and then it failed on 2020-06-03 after the update to v6.2. The reads I have provided were used to assemble the contigs they were mapped to in this issue, as well as other assemblies, mappings, and successful anvi-profiles previously.

I used BBMap:

bbmap.sh in1=Troubl_R1.qc.fq.gz in2=Troubl_R2.qc.fq.gz out=TroublMap.bam ref=Conts.fa minid=0.9 idfilter=0.97 lhist=TroublMap.ReadLeng.txt ihist=TroublMap.InsertLeng.txt statsfile=TroublMap.Stats.txt threads=4 nodisk

This and more mapping information are here: https://github.com/Gsmith535/AnvioError_200605/

For quick reference, the interpretation by BBMap was (from TroublMap.err.txt):

java -Djava.library.path=/usr/local/bioinfo/bbmap/jni/ -ea -Xmx213975m -cp /usr/local/bioinfo/bbmap/current/ align2.BBMap build=1 overwrite=true fastareadlen=500 in1=Troubl_R1.qc.fq.gz in2=Troubl_R2.qc.fq.gz out=TroublMap.bam ref=Conts.fa minid=0.9 idfilter=0.97 lhist=TroublMap.ReadLeng.txt ihist=TroublMap.InsertLeng.txt statsfile=TroublMap.Stats.txt threads=4 nodisk

ekiefl commented 4 years ago

Thanks for this info. @meren may have some insights into this mapping strategy--I don't. But I will add that I find it unsurprising that this did not fail in v6.1. v6.2 was released primarily to introduce a new algorithm and implementation of anvi-profile that is more accurate and faster, if not without hiccups. Sorry that you have fallen victim to one of them.

ekiefl commented 4 years ago

The issue seems fixed. See above commit.

Also, introduced in 76a76942ced606349eada233c44ce74f0587035d, all errors in any of the threads are propagated up to the main thread and raised to avoid this hanging indefinitely. Good riddance.

Thanks for the report @Gsmith535. To experience this fix, you can continue trying to install master, or hang around for v6.3.

Gsmith535 commented 4 years ago

I wanted to follow up for more info, if possible.

For full disclosure, I did notice that my mapping command included all alignments, not only the "good" alignments (out= vs outm= , oh the joys of copy/paste and not catching all of the necessary modifications). Our cluster no longer has Anvio v6.2 so I cannot test myself, but would you want a bam that only had the "good" alignments to test?

There were numerous other unexpected (e.g. very long) alignments in the bam.

samtools view TroublMapSort.bam | awk -F '\t' '{ if ($9 >= 10000) { print $4 " " $5 " " $8 " " $9 } }'
(Returns many alignments with template length >10,000, which seems far from ideal with 300 bp reads and mapping qualities ranging from <10 to >25)

I am curious how Anvio handles these - does it ignore them, or does it force them? What is Anvio v6.2 doing differently form v6.1 that elicits the hanging?

I assume that this isn't a priority, so I completely understand slow or no response.

Thank you for the help! I am glad I could "contribute". Cheers @ekiefl !

elizabethmcd commented 4 years ago

Hi @Gsmith535, not sure if you fully resolved the above issue, but wanted to add my experience. I found this issue because I also had the same error thrown in anvi-profile (v6.2) for some BAM files that I had generated mapping from BBMap. Anecdotally within our lab, we've had a lot of issues with BBMap with misalignments or things that just don't make sense and we don't have an explanation for them. Once I remapped with bowtie2 for each of my samples to the coassembly, I didn't get this error. I've also had problems with other programs feeding in the BAM output from BBMap, as it seems to format it differently than bowtie2 in addition to weird alignment issues. For mapping I have mostly switched to minimap2 or bowtie2 since I've had several issues with BBMap overall. Just wanted to add this input in case anybody else happens upon this issue.

Gsmith535 commented 4 years ago

Hi @elizabethmcd. Thank you for the information about minimap and bowtie integrating better with Anvi'o in general. I will try these other options in the future if the issue remains in future datasets and analyses.

merenlab / anvio

During anvi-profile Config Error: Read.trim leads to indefinite hanging #1436