samtools mpileup: -B or not to -B?

[yenhuahuang1]

Hi,

I was using samtools (0.1.12a) mpileup and found that with the suggested default parameters as

samtools mpileup -ug -f chrs.fa -r 1:1-100000 s_5_6_aligned.sorted.bam | bcftools view -cv -

I just got no useful variants:

[mpileup] 1 samples in 1 input files
<mpileup> Set max per-sample depth to 8000
##fileformat=VCFv4.0
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT s_5_6_aligned.sorted.bam
[afs] 0:0.000 1:0.000 2:0.000

However, with -B in the parameter as

samtools mpileup -Bug -f chrs.fa -r 1:1-100000 s_5_6_aligned.sorted.bam | bcftools view -cv -

I got output as follows

#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT s_5_6_aligned.sorted.bam
1 10124 . C A 3.02 . DP=1;AF1=0.9999;CI95=0.5,1;DP4=0,0,1,0;MQ=30 PL 30,3,0
1 10127 . A C 3.02 . DP=1;AF1=0.9999;CI95=0.5,1;DP4=0,0,1,0;MQ=30 PL 30,3,0
1 10177 . A C 3.02 . DP=1;AF1=0.9999;CI95=0.5,1;DP4=0,0,1,0;MQ=30 PL 30,3,0
1 10241 . T C 3.02 . DP=1;AF1=0.9999;CI95=0.5,1;DP4=0,0,1,0;MQ=30 PL 30,3,0
1 17489 . A C 3.02 . DP=1;AF1=0.9999;CI95=0.5,1;DP4=0,0,0,1;MQ=30 PL 30,3,0
1 20132 . T G 3.02 . DP=1;AF1=0.9999;CI95=0.5,1;DP4=0,0,1,0;MQ=30 PL 30,3,0
1 69511 . A G 99 . DP=37;AF1=1;CI95=1,1;DP4=0,0,11,26;MQ=26 PL 255,111,0
1 75933 . A C 3.02 . DP=1;AF1=0.9999;CI95=0.5,1;DP4=0,0,0,1;MQ=30 PL 30,3,0
1 88318 . A C 3.02 . DP=1;AF1=0.9999;CI95=0.5,1;DP4=0,0,0,1;MQ=30 PL 30,3,0
1 89923 . A C 3.02 . DP=1;AF1=0.9999;CI95=0.5,1;DP4=0,0,1,0;MQ=30 PL 30,3,0
1 89925 . T G 3.02 . DP=1;AF1=0.9999;CI95=0.5,1;DP4=0,0,1,0;MQ=30 PL 30,3,0
[afs] 0:17934.984 1:9.871 2:6.145

Questions:
1. Does this mean that the seq qual (or depth) of my reads is low?
2. Any other parameters for mpileup should be tried?

Many thanks.

[yenhuahuang1]

[follow up!] partially resolved by using bwa, but not soap2

I just noticed that this bam file I have prepared did not contain mapping information such as "XT:A:U *X0:i:1 *X1:i:5 *XM:i:0 *XO:i:0 *XG:i:0".

This bam file was generated by using soap2sam.pl and then samtools view ...

Now I switch to bwa and mapping information is now complete (containing XT:A:U *X0:i:1 *X1:i:5 *XM:i:0 *XO:i:0 *XG:i:0, etc.). Using samtools mpileup with its default parameters and piping the output to bcftools can show variant information.

Does this mean that samtools mpileup use such mapping information to dump variants?

[sergiodealencar]

Hi,

I was using samtools (0.1.12a) mpileup and found that with the suggested default parameters as

samtools mpileup -ug -f chrs.fa -r 1:1-100000 s_5_6_aligned.sorted.bam | bcftools view -cv -

I just got no useful variants:

[mpileup] 1 samples in 1 input files
<mpileup> Set max per-sample depth to 8000
##fileformat=VCFv4.0
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT s_5_6_aligned.sorted.bam
[afs] 0:0.000 1:0.000 2:0.000

However, with -B in the parameter as

samtools mpileup -Bug -f chrs.fa -r 1:1-100000 s_5_6_aligned.sorted.bam | bcftools view -cv -

I got output as follows

#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT s_5_6_aligned.sorted.bam
1 10124 . C A 3.02 . DP=1;AF1=0.9999;CI95=0.5,1;DP4=0,0,1,0;MQ=30 PL 30,3,0
1 10127 . A C 3.02 . DP=1;AF1=0.9999;CI95=0.5,1;DP4=0,0,1,0;MQ=30 PL 30,3,0
1 10177 . A C 3.02 . DP=1;AF1=0.9999;CI95=0.5,1;DP4=0,0,1,0;MQ=30 PL 30,3,0
1 10241 . T C 3.02 . DP=1;AF1=0.9999;CI95=0.5,1;DP4=0,0,1,0;MQ=30 PL 30,3,0
1 17489 . A C 3.02 . DP=1;AF1=0.9999;CI95=0.5,1;DP4=0,0,0,1;MQ=30 PL 30,3,0
1 20132 . T G 3.02 . DP=1;AF1=0.9999;CI95=0.5,1;DP4=0,0,1,0;MQ=30 PL 30,3,0
1 69511 . A G 99 . DP=37;AF1=1;CI95=1,1;DP4=0,0,11,26;MQ=26 PL 255,111,0
1 75933 . A C 3.02 . DP=1;AF1=0.9999;CI95=0.5,1;DP4=0,0,0,1;MQ=30 PL 30,3,0
1 88318 . A C 3.02 . DP=1;AF1=0.9999;CI95=0.5,1;DP4=0,0,0,1;MQ=30 PL 30,3,0
1 89923 . A C 3.02 . DP=1;AF1=0.9999;CI95=0.5,1;DP4=0,0,1,0;MQ=30 PL 30,3,0
1 89925 . T G 3.02 . DP=1;AF1=0.9999;CI95=0.5,1;DP4=0,0,1,0;MQ=30 PL 30,3,0
[afs] 0:17934.984 1:9.871 2:6.145

Questions:
1. Does this mean that the seq qual (or depth) of my reads is low?
2. Any other parameters for mpileup should be tried?

Many thanks.

Hi yenhuahuang1,

The read depth (DP) shown in your output is indeed low, with most of them being equal to 1.

Cheers,
Sérgio

[kissthefuture]

Hi yenhuahuang1,

The read depth (DP) shown in your output is indeed low, with most of them being equal to 1.

Cheers,
Sérgio

You can see the reply in this page http://seqanswers.com/forums/showthread.php?t=8786.
And you can try samtools mpileup -B

[maize]

snp calling with samtools in transcriptome data

hi all,
i am a new for snp calling with samtools in transcriptome data. recently, i am trying to call snps with samtools. I set the option as follows:
samtools mpileup -B -ugSD -f ref.fa aln_sorted.bam | bcftools view -Nbcvg - > aa.bcf
i use the default that samtools manual lists.but i think i need set the option,especially for -D(such as -D100), according to my data, but i don't know the rules or criterion clearly. i think the -D is difficult to set because the data is from RNA-seq. so i am not dry behind the ears for this.
can you help me, dear? thx~