Samtools mpileup, coverage and view provide different read count

Question

[george]

I want to get the number of each covering each position in the genome using samtools. I used samtools depth, mpileup and view and they all give different results. I tried with different versions of samtools (0.1.1 and 1.6) and using different options, but the results don't match. Is there something that I am missing?

samtools depth file.bam -aa -q 0 -Q 0 | head

chr 1   1083
chr 2   1349
chr 3   1485
chr 4   1669
chr 5   1789
chr 6   1834
chr 7   2137
chr 8   2318
chr 9   2402
chr 10  2762

samtools mpileup file.com -aa -q 0 -Q 0 -f genome.fas | head | cut -f1,2,4

chr 1   T   1081
chr 2   T   1347
chr 3   A   1483
chr 4   A   1667
chr 5   A   1787
chr 6   A   1832
chr 7   C   2135
chr 8   A   2316
chr 9   G   2410
chr 10  C   2765

samtools view file.bam chr:0-1 -c

2947

Answer 1

[Pierre Lindenbaum]

bam2depth.c skips/ignores some reads:https://github.com/samtools/samtools/blob/3a7d0f02e8693620014cb59aea17028c5ab427e2/bam2depth.c#L65

if ( b->core.flag & (BAM_FUNMAP | BAM_FSECONDARY | BAM_FQCFAIL | BAM_FDUP) ) continue;
if ( (int)b->core.qual < aux->min_mapQ ) continue;
if ( aux->min_len && bam_cigar2qlen(b->core.n_cigar, bam_get_cigar(b)) < aux->min_len ) continue;

[george]

What about the difference between depth and mpileup. These two use similar/same functions and you expect those results to be the same

[Pierre Lindenbaum]

not the same filters, you will find things related to PROPER_PAIR : https://github.com/samtools/samtools/blob/master/bam_plcmd.c#L282

 else if ((ma->conf->flag&MPLP_NO_ORPHAN) && (b->core.flag&BAM_FPAIRED) && !(b->core.flag&BAM_FPROPER_PAIR)) skip = 1;

see all under: https://github.com/samtools/samtools/blob/master/bam_plcmd.c#L229