SAMTOOLS mpileup issues with TCGA data
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6.3 years ago
Vanish007 ▴ 40

Hi everyone,

Thanks for taking the time to read my question. Apologies in advance if I'm asking a basic question.

I'm currently trying to run Samtools mpileup and varscan on TCGA BAM files using the reference FASTA hg19.

samtools mpileup -f Homo_sapiens_assembly19.fasta Normal.rmdup.BAM Tumor.rmdup.BAM > Norm-Tum.mpileup


However I get a strange output after running the mpileup. Here is what the "head" command shows me for my .mpileup file:

1   10003   A   1   ^!. .   0   *   *
1   10004   C   1   .   <   0   *   *
1   10005   C   1   .   <   0
1   10006   C   1   .   <   0
1   10007   T   1   .   <   1   .   A
1   10008   A   2   ..  <3  1   .   =
1   10009   A   2   ..  <3  1   .   B
1   10010   C   2   ..  <3  3   .^/.^>. DAF
1   10011   C   2   ..  <3  3   ... EBE
1   10012   C   2   ..  <3  3   T.. CEF


Varscan2 completes almost as soon as I execute it and I'm left with only the headers with nothing in them: chrom chr_start chr_stop num_positions normal_depth tumor_depth log2_ratio gc_content

Any idea on what could be causing this? I'm currently using "Homo_sapiens_assembly19.fasta" as my reference. I also wanted to note that the BAM file doesn't seem to have the "chr" notation, instead only uses "SN:1, SN:2, etc". Would I have to change the chr notation in my BAM file for mpileup to work?

Apologies for the long post and thanks for reading.

next-gen sequencing sequence alignment • 1.6k views