Hello Bio Stars,

I have doubt regarding duplicate removal from BAM file. I used two tools "samtools rmdup" and Piccard MarkDuplicates.

I would like to understand why both of the tools remove different amount of duplicate reads.

The document says:

The MarkDuplicates tool works by comparing sequences in the 5 prime positions of both reads and read-pairs in a SAM/BAM file

Samtools rmdup: if multiple read pairs have identical external coordinates, only retain the pair with highest mapping quality. In the paired-end mode, this command ONLY works with FR orientation and requires ISIZE is correctly set

Before removing duplicates

57757837 + 0 in total (QC-passed reads + QC-failed reads)
3902505 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
57757837 + 0 mapped (100.00% : N/A)
53855332 + 0 paired in sequencing
27555734 + 0 read1
26299598 + 0 read2
44132336 + 0 properly paired (81.95% : N/A)
45979358 + 0 with itself and mate mapped
7875974 + 0 singletons (14.62% : N/A)
278406 + 0 with mate mapped to a different chr
123930 + 0 with mate mapped to a different chr (mapQ>=5)

Samtools rmdup result

command: Samtools -S input.bam output.bam

17595767 + 0 in total (QC-passed reads + QC-failed reads)
1161712 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
17595767 + 0 mapped (100.00% : N/A)
16434055 + 0 paired in sequencing
8398895 + 0 read1
8035160 + 0 read2
14057950 + 0 properly paired (85.54% : N/A)
14586355 + 0 with itself and mate mapped
1847700 + 0 singletons (11.24% : N/A)
75278 + 0 with mate mapped to a different chr
38992 + 0 with mate mapped to a different chr (mapQ>=5)

Piccard MarkDuplicates result

command: java -jar /apps/picard.jar MarkDuplicates I=input.bam O=outpu.bam M=marked_dup_metrics.txt REMOVE_DUPLICATES=true

41909982 + 0 in total (QC-passed reads + QC-failed reads)
3902505 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
41909982 + 0 mapped (100.00% : N/A)
38007477 + 0 paired in sequencing
19124624 + 0 read1
18882853 + 0 read2
34855826 + 0 properly paired (91.71% : N/A)
36456720 + 0 with itself and mate mapped
1550757 + 0 singletons (4.08% : N/A)
244146 + 0 with mate mapped to a different chr
107980 + 0 with mate mapped to a different chr (mapQ>=5)