Question: varscan.v2.3.9 CNV call "Error: Invalid format or not enough samples in mpileup"
gravatar for pdehoff
2.4 years ago by
pdehoff0 wrote:

I'm hitting a wall and need some help with this issue.

Overview: I have NGS data for a human tumor cell line (Onco_comb_rd.bam) for which I need to identify CNVs compared to a wild type human genome sequence. I understand I need a tumor/normal comparison for samtools mpileup | varscan copynumber (I am perfectly willing to use bcftools cnv if someone can tell me what is supposed to go after the -c and -s options)

I have mapped, sorted, and rmdup'd the tumor data. I do not have a "normal" set of data, so I generated a synthetic data set using one of the bbmap related tools. For the synthetic set, I tried to match the # of tumor reads as well as the approximate insert size. Mapped etc (hg38_synth.rd.bam)

It took a while to get mpileup to function without a memory error, but I eventually got it to work. I have tried this whoel process without piping the data, and with (latest command below), but keep running into the same (or close) error:

[mpileup] 2 samples in 2 input files
Min coverage:   10
Min avg qual:   15
P-value thresh: 0.01
<mpileup> Set max per-file depth to 4000
Reading input from STDIN
Reading mpileup input...
Error: Invalid format or not enough samples in mpileup: ##fileformat=VCFv4.2

At any rate, this is the latest version of the command:

samtools mpileup -Avuf /oasis/projects/nsf/csd414/pdehoff/Reference/GRCh38_latest_genomic.fna hg38_synth.rd.bam Onco_comb_rd.bam | java -Xmx4G -jar /oasis/projects/nsf/csd414/pdehoff/programs/VarScan.v2.3.9.jar copynumber --mpileup Onco_hg38

I've been pulling my hair out over this for a while and I need to understand what I'm doing wrong here. If any of you folks have some suggestions, please let me know. Thank you.

varscan copy number • 761 views
ADD COMMENTlink modified 2.4 years ago • written 2.4 years ago by pdehoff0
gravatar for ATpoint
2.4 years ago by
ATpoint40k wrote:

Try upgrading to the latest version 2.4.3. There have been some changes made in the 2.4.X versions to better handle the mpileup output.

ADD COMMENTlink written 2.4 years ago by ATpoint40k
gravatar for pdehoff
2.4 years ago by
pdehoff0 wrote:

I tried this out and it worked. Initially had the "Parsing Exception on line:" error.

Hopefully this will help other people out. Note that all of what is in the command may or may not be need, but the "awk" part (which I got from somewhere on SourceForge, was key.

samtools mpileup -B -q 1 -f /oasis/projects/nsf/csd414/pdehoff/Reference/GRCh38_latest_genomic.fna hg38_synth.rd.bam Onco_comb_rd.bam | awk 'NF==9 && $4!=0' | java -Xmx4G -jar /oasis/projects/nsf/csd414/pdehoff/programs/VarScan.v2.3.9.jar copynumber varScan --mpileup 1

ADD COMMENTlink written 2.4 years ago by pdehoff0
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