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Cannot access BAM file #8

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tmatsu24 opened this issue Dec 23, 2021 · 13 comments
Open

Cannot access BAM file #8

tmatsu24 opened this issue Dec 23, 2021 · 13 comments

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@tmatsu24
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Hi.
I tired to run MoCaSeq test pipeline several times but every time FILE ERROR happend.

---- Matched BAM-files? ----
Thu Dec 23 12:34:20 UTC 2021 timestamp: 1640262860
+------------+
| FILE ERROR |
+------------+
Cannot access BAM file (/var/pipeline/MoCaSeq_Test/results/bam/MoCaSeq_Test.Tumor.bam).
It either does not exist or is not readable.

Could you help me about this problem ?

@NikdAK
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NikdAK commented Dec 23, 2021

Hi,
please provide us the command you used to call MoCaSeq as well as the full report file located here: MoCaSeq_Test/results/QC/MoCaSeq_Test.report.txt

Additionally the test files are stored with Git LFS, so maybe the download did not work. Could you please check if the files within the test folder are ~50MB?

@tmatsu24
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tmatsu24 commented Dec 23, 2021 via email

@NikdAK
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NikdAK commented Dec 27, 2021

So the issue here is, that the reference files are not downloaded as they should be (your ref/ folder is probably empty).

Let's try to fix this:
First enter the docker ennvironment using this:

docker run \
-it --entrypoint=/bin/bash \
-v ${working_directory}:/var/pipeline/ \
rolandradlab/mocaseq:0.4.53

You are now inside the docker container with all the tools installed and code available.

Now executing the following lines:

rm -r temp/ ref/
mkdir temp/
Preparation_GetReferenceDataMouse.sh config-file-from-git-folder temp/ 2>&1 | tee GetReferenceLog.txt

This should start the download and indexing of the needed reference files (this takes a while).
This is working for me right now, but sent me the log file in case something goes wrong.

After that can exit the container using the "exit" command, move the reference files somewhere save and always mount it like this:

docker run
-v ${working_directory}:/var/pipeline/
-v ${ref_directory}:/var/pipeline/ref/
rolandradlab/mocaseq:0.4.53
--test yes

Hope this helps.

@tmatsu24
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tmatsu24 commented Dec 31, 2021 via email

@NikdAK
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NikdAK commented Jan 4, 2022

Looks like the download of the FASTA file is not working for some reason, maybe permission or unstable connection issues?

Try the following individual commands from the script:
wget -nv ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/001/635/GCA_000001635.8_GRCm38.p6/GCA_000001635.8_GRCm38.p6_genomic.fna.gz

There should be a 860,9 MB .gz file now and then 2.9 GB after decompressing.

gunzip GCA_000001635.8_GRCm38.p6_genomic.fna.gz

@tmatsu24
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tmatsu24 commented Jan 6, 2022 via email

@tmatsu24
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tmatsu24 commented Jan 6, 2022 via email

@NikdAK
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NikdAK commented Jan 7, 2022

So now the problem is this:
[bwa_index] Update BWT... Killed

Apparently something in your system killed the BWA indexing process. Are you running on a system with restrictions on runtime or memory etc.?
I recommend you run this script line-by-line manually to see what is going on.

@tmatsu24
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tmatsu24 commented Jan 8, 2022 via email

@NikdAK
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NikdAK commented Jan 10, 2022

32GB should be more than enough for BWA. But after managing the references, you should also change the default (32GB) of the MoCaSeq pipeline using -r 15 and probably also the number of threads with -t.

So again, I can not reproduce this error, so you will have to go into a script and execute line-by-line to find the exact error location (the FASTA should already be there and processed by the other script).
The script you need to go through is: Preparation_GenerateBWAIndex.sh (check to repository folder in this repo)

You need the following variables:

VersionMouse=GRCm38.p6
config_file=/opt/MoCaSeq/config.sh
species=Mouse

@tmatsu24
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tmatsu24 commented Jan 14, 2022 via email

@NikdAK
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NikdAK commented Jan 17, 2022

Could you please provide me the entire S821-WES/QC/S821-WES.report.txt file?
Please attach it as a file, do not copy the raw text in here.

@tmatsu24
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Hi,

Actually, I tried again and this time "Cannot access BAM file (/var/pipeline/S821-WES/results/bam/S821-WES.Tumor.bam)." error was not occured but other error seems to be happen (I've ovrewritten the first run of S821-WES.report.txt....).

Also, I thought "--test run" worked without problem but several error seems to be happen.

I attach "MoCaSeq_Test.report.txt" and 2nd time run of "S821-WES.report.txt"

Thaks in advance
MoCaSeq_Test.report.txt
S821-WES.report.txt
.

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