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CGATOxford / UMI-tools

Tools for handling Unique Molecular Identifiers in NGS data sets
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nosetests showing 1 error in python3 (and multiple for python 2.7 with appropriate umi tools version) #450

Closed alexander-e-f-smith closed 6 months ago

alexander-e-f-smith commented 3 years ago

Hi I've not been able to get a completely error free output after new installation and running your nose tests. See below for details of how I have run this and environment set-up before hand. I get 1 error around whitelisting for python3 set-up when running nose tests. And multiple errors for old installation of umi tools (0.5.1) in python 2.7....although i'm not sure if the nose tests are appropriate here (nosetest umi_test.py is aligned for the installation version of umi_tools. See below for details:

In python 3.7 export PYTHONHASHSEED=0

root@feac0438ff76:/usr/umitools# which umi_tools /opt/venv/bin/umi_tools

root@feac0438ff76:/usr/umitools# umi_tools --version UMI-tools version: 1.1.1

root@e83a97661f88:/usr/umitools/UMI-tools-1.1.1# nosetests tests/test_umi_tools.py

OUTPUT: 

../usr/umitools/UMI-tools-1.1.1/tests/test_umi_tools.py:240: YAMLLoadWarning: calling yaml.load() without Loader=... is deprecated, as the default Loader is unsafe. Please read https://msg.pyyaml.org/load for full details.
  tool_tests = yaml.load(open(fn))
......F.................................................
======================================================================
FAIL: umi_tools.py/whitelist_scrb_seq
----------------------------------------------------------------------
Traceback (most recent call last):
  File "/opt/venv/lib/python3.7/site-packages/nose/case.py", line 198, in runTest
    self.test(*self.arg)
  File "/usr/umitools/UMI-tools-1.1.1/tests/test_umi_tools.py", line 202, in check_script
    ok_(not fail, msg)
AssertionError: files /tmp/tmp17lqxxuh/stdout and tests/whitelist_umi_output.sam are not the same
/bin/bash -c 'umi_tools dedup --paired --log=test.log --filter-umi --umi-whitelist=/usr/umitools/UMI-tools-1.1.1/tests/umi_whitelist.tsv --umi-whitelist-paired=/usr/umitools/UMI-tools-1.1.1/tests/umi_whitelist.tsv --out-sam --random-seed=123456789 --stdin=/usr/umitools/UMI-tools-1.1.1/tests/whitelist_umi_input.bam |sort > /tmp/tmp17lqxxuh/stdout'
md5: output=1531, ed9d1ea5c354335f290837080ffb1115 reference=1531, 4d05033cdda6ca1bbbfa412409247d42first 10 differences: @PG    ID:hisat2   PN:hisat2   VN:2.1.0    CL:"/home/PROT-FILESVR1/proteomics/tss38/cgat-install/conda-install/envs/py36-v1/bin/hisat2-align-s --wrapper basic-0 --threads 1 --rna-strandness RF -x /home/PROT-FILESVR1/proteomics/tss38/references/genomes/hisat2/hg38 --known-splicesite-infile geneset.dir/geneset_all.junctions --novel-splicesite-outfile hisat_no_filter.dir/CL-1-1.hisat.bam_novel_junctions -1 /tmp/10587.inpipe1 -2 /tmp/10587.inpipe2"
NS500105:308:HCGGHAFXY:1:11101:20827:5614_CTCAGTCTTTCGTTCG  99  chr1    22053   60  1S67M   =   192659  170684  TGGGCTGCCCACAGGGCTCCTCAGTCTAAGCCAAGTGGTGTGTCATAGTCCCCTGGCCCCAGTAATGG    /EEEEEEEEEEEEEAEEEAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEEEEE    AS:i:-11    ZS:i:-11    XN:i:0  XM:i:2  XO:i:0  XG:i:0  NM:i:2  MD:Z:13A46T6    YS:i:-1 YT:Z:CP XS:A:-  NH:i:1
--
@SQ SN:chr1 LN:248956422
NS500105:308:HCGGHAFXY:1:11101:25537:9008_GTTGTCGAAGACACTC  99  chr1    19796   1   1S65M   =   190403  170684  CCCATCTTTTCTGGCTGGGGAGAGGCCTTCATCTGCTGTAAAGGGTCCTCCAGCACAAGCTGTCTT  /EEAEEEEEEEEEEEEEEEEEEEEEE/EEEEEEEEEEEEAEEEEEEEEEEEAEEEEEEEEEEEEEE  AS:i:-1 ZS:i:-1 XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:65 YS:i:-2 YT:Z:CP XS:A:-  NH:i:4
--
@SQ SN:chr10    LN:133797422
NS500105:308:HCGGHAFXY:1:11101:8596:6657_GGATAACGAATTCCGG   355 chr1    91436   1   68M =   91623   255 GCAGAGGTCAGCAAGGCAAACCCGAGCCCAGGGATGCGGGGTGGGGGCAGGTACATCCTCTCTTGAGC    6EEEEEEEEEEEA/EEEAEEEEEEEEEEEEE/EEEEEEEEEAEEEEEEEEEEE6EE6E/EEEEEE/E<    AS:i:0  ZS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:68 YS:i:-1 YT:Z:CP XS:A:-  NH:i:3
--
@SQ SN:chr10_GL383545v1_alt LN:179254
NS500105:308:HCGGHAFXY:1:11101:8596:6657_GGATAACGAATTCCGG   403 chr1    91623   1   7M461N60M1S =   91436   -255    CCAAACTCTTGGTTGTGTTCTTTGATTAGTGCCTGTGACGCAGCTTCAGGAGGTCCTGAGAACGTGTAEE/AA</E//<///A//</E666//666A66EE/A66EE6E6AE//AAA6A/66E/666A6EEA6A</    AS:i:-1 ZS:i:-1 XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:67 YS:i:0  YT:Z:CP XS:A:-  NH:i:3
--
@SQ SN:chr10_GL383546v1_alt LN:309802
NS500105:308:HCGGHAFXY:1:11101:8596:6657_GGATAACGAATTCCGG   99  chr1    91436   1   68M =   268198  176898  GCAGAGGTCAGCAAGGCAAACCCGAGCCCAGGGATGCGGGGTGGGGGCAGGTACATCCTCTCTTGAGC    6EEEEEEEEEEEA/EEEAEEEEEEEEEEEEE/EEEEEEEEEAEEEEEEEEEEE6EE6E/EEEEEE/E<    AS:i:0  ZS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:68 YS:i:-1 YT:Z:CP XS:A:-  NH:i:3
--
@SQ SN:chr10_KI270824v1_alt LN:181496
NS500105:308:HCGGHAFXY:1:11102:25272:14512_GTTCTGCTCTGAGTGT 99  chr1    21354   60  68M =   191926  170649  TCCTGACTACAATAACAGATTCTGGGTGTCCCTGGCATCCACTCTCTCTCCCTTATTATCCCTTCAGT    E</E6EAEEEEE/EE/E//EEEA//AAAE/<A///EEA//EE///<//<//A66//E//AE/A////A    AS:i:-12    ZS:i:-12    XN:i:0  XM:i:4  XO:i:0  XG:i:0  NM:i:4  MD:Z:18C35C2G7C2    YS:i:-1 YT:Z:CP XS:A:-  NH:i:1
--
@SQ SN:chr10_KI270825v1_alt LN:188315
NS500105:308:HCGGHAFXY:1:11103:19873:1262_TGAGAGTGGAAGTGCA  355 chr1    18999   1   1S65M   =   19051   121 TCTTGCATCTCATGGAACGCCATTTCCCCAGACATCCCTGTGGCTGGCTCCTGATGCCCGAGGCCC  AEEEEE/EEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEEEEEEEEEEE<EEEEEEEEEEEEEAE  AS:i:-1 ZS:i:-1 XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:65 YS:i:-1 YT:Z:CP XS:A:-  NH:i:5
--
@SQ SN:chr11    LN:135086622
NS500105:308:HCGGHAFXY:1:11103:19873:1262_TGAGAGTGGAAGTGCA  403 chr1    19051   1   67M1S   =   18999   -121    ATGCCCGAGGCCCAAGTGTCTGATGCTTTAAGGCACATCACCCCACTCATGCTTTTCCATGTTCTTTA    EEEEEEEEEEEEEEEAEEAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAE/    AS:i:-1 ZS:i:-1 XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:67 YS:i:-1 YT:Z:CP XS:A:-  NH:i:5
--
@SQ SN:chr11_GL383547v1_alt LN:154407
NS500105:308:HCGGHAFXY:1:11103:8261:10925_ATCCATGGTCGTAGGT  355 chr1    14224   0   5S63M   =   184948  170804  GCCCGTCTGCAACAGCTGCCCCTGCTGACGGCCCTTCTCTCCTCCCTCTCATCCCAGAGAAACAGGTC    /EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEEAEEEEEEEEEEEEEEEEE    AS:i:-10    ZS:i:-10    XN:i:0  XM:i:1  XO:i:0  XG:i:0  NM:i:1  MD:Z:24T38  YS:i:-10    YT:Z:CP XS:A:-  NH:i:2
--
@SQ SN:chr11_JH159136v1_alt LN:200998
NS500105:308:HCGGHAFXY:1:11104:19418:19384_GTTCACCTAGTCGAGA 355 chr1    17033   1   4S64M   =   17218   257 CGTTACATAGAAGTAGTTCTCTGGGACCTGCAAGATTAGGCAGGGACATGTGAGAGGTGACAGGGACC    /EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEEEEEEAEEEEEEEE    AS:i:-4 ZS:i:-4 XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:64 YS:i:0  YT:Z:CP XS:A:-  NH:i:5
--
@SQ SN:chr11_JH159137v1_alt LN:191409
NS500105:308:HCGGHAFXY:1:11104:19418:19384_GTTCACCTAGTCGAGA 403 chr1    17218   1   68M =   17033   -257    TCCCACCCCTCCCACCTGCTGTTCCAGCTGCTCTCTCTTGCTGATGGACAAGGGGGCATCAAACAGCT    EEEEEEEEEEEAE/EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE</    AS:i:0  ZS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:68 YS:i:-4 YT:Z:CP XS:A:-  NH:i:5

----------------------------------------------------------------------
Ran 58 tests in 97.134s

FAILED (failures=1)

I also ran this on older version of UMI_tools in python 2.7 (version 0.5.1), which gave may more errors using same test conditions. Although I am not sure whether this is valid in python2.7 at all. 

In Python2.7

export PYTHONHASHSEED=0

nosetests tests/test_umi_tools.py (extract of output below)

FAIL: umi_tools.py/whitelist_scrb_seq
----------------------------------------------------------------------
Traceback (most recent call last):
  File "/usr/local/lib/python2.7/dist-packages/nose/case.py", line 197, in runTest
    self.test(*self.arg)
  File "/tmp/UMI-tools/tests/test_umi_tools.py", line 202, in check_script
    ok_(not fail, msg)
AssertionError: files /tmp/tmplFA85o/stdout and tests/group_unsorted.sam are not the same
/bin/bash -c 'umi_tools group -L test.log --out-sam --random-seed=123456789 --method=directional --no-sort-output  --output-bam --stdin=/tmp/UMI-tools/tests/chr19.bam |sort > /tmp/tmplFA85o/stdout'
md5: output=55221, fb36cbc9c92278137833599787af76ba reference=55221, 793edea471eb96ad61cf38d5b8d916f4first 10 differences: @SQ  SN:chr1 LN:197195432
@SQ SN:chr10    LN:129993255
--
@SQ SN:chr10    LN:129993255
@SQ SN:chr11    LN:121843856
--
@SQ SN:chr11    LN:121843856
@SQ SN:chr12    LN:121257530
………………………………………..
……………………………………….
Ran 35 tests in 55.604s

FAILED (failures=16)
TomSmithCGAT commented 3 years ago

Hi. For the python 3.7 test, can you try running export PYTHONHASHSEED=0 before nosetests. See https://github.com/CGATOxford/UMI-tools/issues/440 for a related outstanding issue.

alexander-e-f-smith commented 3 years ago

Hi, thanks for the response. That's how I have run it (see previous post details...I have pasted in the relative environment at the top.....including PYTHONHASHSEED=0). Best

TomSmithCGAT commented 3 years ago

Very sorry. Yes, you clearly mentioned that...

TomSmithCGAT commented 3 years ago

Hmm... can't reproduce with clean conda or pip installation.

How are you installing?

What do you get when you run the command manually? umi_tools dedup --paired --log=test.log --filter-umi --umi-whitelist=/usr/umitools/UMI-tools-1.1.1/tests/umi_whitelist.tsv --umi-whitelist-paired=/usr/umitools/UMI-tools-1.1.1/tests/umi_whitelist.tsv --out-sam --random-seed=123456789 --stdin=/usr/umitools/UMI-tools-1.1.1/tests/whitelist_umi_input.bam |sort > tmp

alexander-e-f-smith commented 3 years ago

Hi See attached for output (no error seemingly). I have installed inside docker. I have now tried either inside a venv or using system installed python 3 and get same output to your code below. Here is installation routine part of Dockerfile which gives no error that I can see (I remove python 3.5 from requirements.txt as I have python 3.7 installed):

RUN apt-get update && \ wget https://github.com/CGATOxford/UMI-tools/archive/1.1.1.zip && \ unzip 1.1.1.zip && \ cd /usr/umitools/UMI-tools-1.1.1 && \ sed -e "s/python>=3.5//g" -i requirements.txt && \ pip3 install -r requirements.txt && \ python3 setup.py install

From: Tom Smith notifications@github.com Sent: 07 January 2021 14:37 To: CGATOxford/UMI-tools UMI-tools@noreply.github.com Cc: Smith, Alexander alexander.e.smith@kcl.ac.uk; Author author@noreply.github.com Subject: Re: [CGATOxford/UMI-tools] nosetests showing 1 error in python3 (and multiple for python 2.7 with appropriate umi tools version) (#450)

Hmm... can't reproduce with clean conda or pip installation.

How are you installing?

What do you get when you run the command manually? umi_tools dedup --paired --log=test.log --filter-umi --umi-whitelist=/usr/umitools/UMI-tools-1.1.1/tests/umi_whitelist.tsv --umi-whitelist-paired=/usr/umitools/UMI-tools-1.1.1/tests/umi_whitelist.tsv --out-sam --random-seed=123456789 --stdin=/usr/umitools/UMI-tools-1.1.1/tests/whitelist_umi_input.bam |sort > tmp

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHubhttps://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fgithub.com%2FCGATOxford%2FUMI-tools%2Fissues%2F450%23issuecomment-756155183&data=04%7C01%7Calexander.e.smith%40kcl.ac.uk%7C06d2c2e7bb4c4792b1c008d8b319ceed%7C8370cf1416f34c16b83c724071654356%7C0%7C0%7C637456270714553054%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=AOHw0lxmPRtN1%2FJUxwskCiHR9K%2B%2FWU%2F%2F1nY6%2FiEgXmQ%3D&reserved=0, or unsubscribehttps://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fgithub.com%2Fnotifications%2Funsubscribe-auth%2FAG3NR5SQWM54BYDTL57FELDSYXBLZANCNFSM4VY4ZLNA&data=04%7C01%7Calexander.e.smith%40kcl.ac.uk%7C06d2c2e7bb4c4792b1c008d8b319ceed%7C8370cf1416f34c16b83c724071654356%7C0%7C0%7C637456270714553054%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=UApTYHFXofNtelQP9QROjnDxsnVd4wIorapneikPGEY%3D&reserved=0.

alexander-e-f-smith commented 3 years ago

btw, are you able to see the attached?

Dr Alexander Smith Dept Haematology King's College NHS trust Denmark Hill London SE5 9RS

From: Smith, Alexander alexander.e.smith@kcl.ac.uk Sent: 07 January 2021 15:01 To: CGATOxford/UMI-tools UMI-tools@noreply.github.com; CGATOxford/UMI-tools reply@reply.github.com Cc: Author author@noreply.github.com Subject: Re: [CGATOxford/UMI-tools] nosetests showing 1 error in python3 (and multiple for python 2.7 with appropriate umi tools version) (#450)

Hi See attached for output (no error seemingly). I have installed inside docker. I have now tried either inside a venv or using system installed python 3 and get same output to your code below. Here is installation routine part of Dockerfile which gives no error that I can see (I remove python 3.5 from requirements.txt as I have python 3.7 installed):

RUN apt-get update && \ wget https://github.com/CGATOxford/UMI-tools/archive/1.1.1.zip && \ unzip 1.1.1.zip && \ cd /usr/umitools/UMI-tools-1.1.1 && \ sed -e "s/python>=3.5//g" -i requirements.txt && \ pip3 install -r requirements.txt && \ python3 setup.py install

From: Tom Smith notifications@github.com Sent: 07 January 2021 14:37 To: CGATOxford/UMI-tools UMI-tools@noreply.github.com Cc: Smith, Alexander alexander.e.smith@kcl.ac.uk; Author author@noreply.github.com Subject: Re: [CGATOxford/UMI-tools] nosetests showing 1 error in python3 (and multiple for python 2.7 with appropriate umi tools version) (#450)

Hmm... can't reproduce with clean conda or pip installation.

How are you installing?

What do you get when you run the command manually? umi_tools dedup --paired --log=test.log --filter-umi --umi-whitelist=/usr/umitools/UMI-tools-1.1.1/tests/umi_whitelist.tsv --umi-whitelist-paired=/usr/umitools/UMI-tools-1.1.1/tests/umi_whitelist.tsv --out-sam --random-seed=123456789 --stdin=/usr/umitools/UMI-tools-1.1.1/tests/whitelist_umi_input.bam |sort > tmp

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IanSudbery commented 3 years ago

No i'm not sure attachments work in github issues.

The problem in the nosetests for the python3 version appears to be that the your umi_tools appears to be outputting a sam file with a header, where as the reference does not contain a header.

I wonder if this is a pysam/samtools version issue. Has a new version of pysam/samtools be released since we last did a rebuild?

alexander-e-f-smith commented 3 years ago

Hi again I think I have a dependency issue. A pip install on my local ubuntu machine (not in docker container) works with no errors from nosetests (python 3.6.9 in venv). I'll investigate further and let you know if I find a culprit...such as pysam. A

From: Ian Sudbery notifications@github.com Sent: 07 January 2021 15:56 To: CGATOxford/UMI-tools UMI-tools@noreply.github.com Cc: Smith, Alexander alexander.e.smith@kcl.ac.uk; Author author@noreply.github.com Subject: Re: [CGATOxford/UMI-tools] nosetests showing 1 error in python3 (and multiple for python 2.7 with appropriate umi tools version) (#450)

No i'm not sure attachments work in github issues.

The problem in the nosetests for the python3 version appears to be that the your umi_tools appears to be outputting a sam file with a header, where as the reference does not contain a header.

I wonder if this is a pysam/samtools version issue. Has a new version of pysam/samtools be released since we last did a rebuild?

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHubhttps://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fgithub.com%2FCGATOxford%2FUMI-tools%2Fissues%2F450%23issuecomment-756204183&data=04%7C01%7Calexander.e.smith%40kcl.ac.uk%7C6607af6314024f0a866a08d8b324d18f%7C8370cf1416f34c16b83c724071654356%7C0%7C0%7C637456318013968398%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=e4UFcEBSLaxRU%2FB9WuNGwi%2B7LB7Wi5ROcmiHsH1kgdU%3D&reserved=0, or unsubscribehttps://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fgithub.com%2Fnotifications%2Funsubscribe-auth%2FAG3NR5TSJADMIIJLQ55JSRTSYXKTLANCNFSM4VY4ZLNA&data=04%7C01%7Calexander.e.smith%40kcl.ac.uk%7C6607af6314024f0a866a08d8b324d18f%7C8370cf1416f34c16b83c724071654356%7C0%7C0%7C637456318013968398%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=Wdbd21OVqZAYzJQBfAnXmXYZCGTsizkC95vgns7cO1E%3D&reserved=0.

alexander-e-f-smith commented 3 years ago

Hi Tom/Ian I think this is a docker-related issue I haven't been able to resolve yet. I cannot get the nosetests to run faultlessly (same single error everytime) for several docker image variants, one of which uses the same ubuntu base (18) and venv environment (same modules installed on same python version) as my host machine on which nosetest runs faultlessly. I also get faultless nosetests results on another machine running ubuntu 20 when not using docker too. For docker, the error is same for docker containers build on ubuntu or debian buster/buster-slim in both python 3.6 and 3.7.

Dr Alexander Smith Dept Haematology King's College NHS trust Denmark Hill London SE5 9RS

From: Smith, Alexander alexander.e.smith@kcl.ac.uk Sent: 07 January 2021 16:51 To: CGATOxford/UMI-tools UMI-tools@noreply.github.com; CGATOxford/UMI-tools reply@reply.github.com Cc: Author author@noreply.github.com Subject: Re: [CGATOxford/UMI-tools] nosetests showing 1 error in python3 (and multiple for python 2.7 with appropriate umi tools version) (#450)

Hi again I think I have a dependency issue. A pip install on my local ubuntu machine (not in docker container) works with no errors from nosetests (python 3.6.9 in venv). I'll investigate further and let you know if I find a culprit...such as pysam. A

From: Ian Sudbery notifications@github.com Sent: 07 January 2021 15:56 To: CGATOxford/UMI-tools UMI-tools@noreply.github.com Cc: Smith, Alexander alexander.e.smith@kcl.ac.uk; Author author@noreply.github.com Subject: Re: [CGATOxford/UMI-tools] nosetests showing 1 error in python3 (and multiple for python 2.7 with appropriate umi tools version) (#450)

No i'm not sure attachments work in github issues.

The problem in the nosetests for the python3 version appears to be that the your umi_tools appears to be outputting a sam file with a header, where as the reference does not contain a header.

I wonder if this is a pysam/samtools version issue. Has a new version of pysam/samtools be released since we last did a rebuild?

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IanSudbery commented 3 years ago

Thats very peculiar. The problem is that one your tests the header is being output, and our reference file doesn't have headers. This doesn't seem like it should be an OS related problem.

TomSmithCGAT commented 6 months ago

Closing due to inactivity