PCR duplicates in RNASeq
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0
Entering edit mode
5.3 years ago
Prakash ★ 2.2k

Hello Bio stars,

I have small query regarding identification and removal of PCR duplicates from RNASeq data. The Tophat2 alignment stats calculated from samtool flagstat shows that there is no PCR duplicates (below alignment stats), but when I used samtools rmdup , it removes significant amount of reads from alignment. What discrepancy is this or I am missing some information.

Alignment stats before removing duplicates

78300010 + 0 in total (QC-passed reads + QC-failed reads)
4330622 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
78300010 + 0 mapped (100.00% : N/A)
73969388 + 0 paired in sequencing
68770674 + 0 properly paired (92.97% : N/A)
71824262 + 0 with itself and mate mapped
2145126 + 0 singletons (2.90% : N/A)
422144 + 0 with mate mapped to a different chr
158018 + 0 with mate mapped to a different chr (mapQ>=5)


Alignment stats after removing duplicates

20391758 + 0 in total (QC-passed reads + QC-failed reads)
1265797 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
20391758 + 0 mapped (100.00% : N/A)
19125961 + 0 paired in sequencing
17932936 + 0 properly paired (93.76% : N/A)
18650095 + 0 with itself and mate mapped
475866 + 0 singletons (2.49% : N/A)
85116 + 0 with mate mapped to a different chr
32276 + 0 with mate mapped to a different chr (mapQ>=5)

RNA-Seq Forum • 2.4k views
3
Entering edit mode
5.3 years ago

tophat2 does not flag duplicates by default. So flagstat ( the name itself indicates, it gives stats on flags in sam/bam file ) does not show any duplicates.

rmdup removes duplicates based on alignment positions. So it removes them irrespective of flagging.

BTW, duplicate removal is not a good idea for RNA-Seq.

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Entering edit mode

Thank you geek_y !

So if rmdup is removing duplicates, it indicate that my sample has duplicates. I understand duplicate removal is not an good idea for differential expression study, but is it not necessary to remove for Alternate splicing analysis.

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Entering edit mode

In RNA-Seq, its difficult to distinguish a true transcript expression from PCR duplicate. Its not advisable to remove duplicates.