varscan2: copynumber detection

[mrfox]

Hi all,

I would like to know if anybody has experience using VarScan2 to call copynumber (assuming we have Tumor/Normal matched sample). Specifically, I want to know how you calculate the parameter "data ratio".

Any suggestion will be appreciated.

[dkoboldt]

Thank you for your message. We developed VarScan and use it for exome-based copy number analysis. The data-ratio value could simply be calculated by looking at the SAMtools "flagstat" for tumor and normal BAM files to calculate the number of reads that are mapped and unique (not marked duplicate). The ratio should be normal OVER tumor:

r = (normal_unique_reads / tumor_unique_reads)

Of course, this assumes that reads are the same length in normal and tumor datasets. If they're not, one would have to adjust for uniquely mapped base pairs.

You may notice, after calling copy number and performing CBS, some samples with a systematic bias in copy number calls where neutral regions seem shifted away from the zero-axis. We believe these to be instances of different exome capture efficiency between samples.

You can normalize raw VarScan copy number values up or down to address these situations; see the VarScan copyCaller command usage for details.

Please feel free to e-mail me directly if you have further questions:
dkoboldt (at) genome [dot] wustl (dot) edu.

[rpauly]

I have a question regarding the mergeSegments.pl that is a part of the copy number workflow from Vrascan.

After a lot of searching, I generated the file with the following columns (Please note, nowhere, on the website is it mentioned that we need the segments.p function from the DNAcopy package to get these columns ):
ID, chrom, loc.start, loc.end, num.mark, seg.mean, bstat, pval, lcl, ucl.

and when I try to run this file with mergeSegments.pl ( perl ../../mergeSegments.pl out.file --output-basename basenametest --verbose 1) it gives me an error:

Use of uninitialized value $input in <HANDLE> at ../../mergeSegments.pl line 446.
readline() on unopened filehandle at ../../mergeSegments.pl line 446.
Can't use an undefined value as a symbol reference at ../../mergeSegments.pl line 456.

What could I change here?