*.bam -> var.bcf No result

[dongshenglulv]

Hi all,

When I tried to call variants by 25 BAM files like this,
$ samtools mpileup -P ILLUMINA -ugf hg19.fasta *.bam | bcftools view -bcvg - > var.raw.bcf ; bcftools view var.raw.bcf | vcfutils.pl varFilter -D 2000 > var.flt.vcf

Then the standard output was
[mpileup] 25 samples in 25 input files
<mpileup> Set max per-file depth to 320
[afs] 0:0.000 1:0.000 2:0.000 3:0.000 4:0.000 5:0.000 6:0.000 7:0.000 8:0.000 9:0.000 10:0.000 11:0.000 12:0.000 13:0.000 14:0.000 15:0.000 16:0.000 17:0.000 18:0.000 19:0.000 20:0.000 21:0.000 22:0.000 23:0.000 24:0.000 25:0.000 26:0.000 27:0.000 28:0.000 29:0.000 30:0.000 31:0.000 32:0.000 33:0.000 34:0.000 35:0.000 36:0.000 37:0.000 38:0.000 39:0.000 40:0.000 41:0.000 42:0.000 43:0.000 44:0.000 45:0.000 46:0.000 47:0.000 48:0.000 49:0.000 50:0.000
and the program just stopped, the VCF file just only had the header information. I don't why, could you help me?

thanks

[DZhang]

Hi,

According to my experience, this usually means that your parameters for SNP calling is not optimal so there is NO SNP called. You may loosen the criteria to try to get some callings, and then tighten them up.

Douglas

https://www.contigexpress.com

[dongshenglulv]

The only parameter for criteria here is -D. I just tried this command,
$ samtools mpileup -P ILLUMINA -ugf hg19.fasta *.bam | bcftools view -cvg - > var.raw.vcf

Then the standard output was the same, and the file var.raw.vcf just had the header information without the body.
Is there any suggestion you can give to me? Thanks very much

[DZhang]

Hi,

I am not entirely sure but I recall there is a step from bcf to vcf. Please check samtools manual, especially the part related to bcftools.

Douglas

https://www.contigexpress.com

[Hkins552]

Have you resolved this problem?

[swbarnes2]

Try running just mpileup to generate a pileup. Does that look normal?

I'm not sure that all those 0's are normal in the output to the terminal.

[Lynette]

I had the same problem as you (had no output from mpileup except headers) from solid paired end data and when I added the -A option I got results!

[swbarnes2]

I had the same problem as you (had no output from mpileup except headers) from solid paired end data and when I added the -A option I got results!

Hmm, real SNPs should be visible without relying solely on anomalous read pairs.

The other thing to try is run mpileup with -B. That will disengage the BAQ calculations. It's possible that you will get more false positives, but I've seen the BAQ calculations hide true positives.

[Lynette]

Bit of background: I'm using solid 5500 data, aligning with bfast, picard remove duplicates, srma (for local-realignment), picard fixmate, and then gatk q-score recalibration. I've been used the -B option and still got no output until I used the -A option

I'm not too if one of those steps is messing up the mate information so I tried picard fixmate after gatk q-score recalibration and still no results from mpileup until I used the -A option. It's very weird when I look at the picard insert size metrics of this sample I have like half (wrong) RF orientation and half (correct) FR orientation.