TopHat extremely low paired mapping rate. PLS HELP!

Felix.Lee · 05-22-2013, 02:00 PM

Hey guys, I have some problems with my paried-end RNA seq analysis on Galaxy. As you can see in the bam flagstat output, my tophat alignment rate is very low.

15620641 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 duplicates
15620641 + 0 mapped (100.00%:-nan%)
15620641 + 0 paired in sequencing
7109757 + 0 read1
8510884 + 0 read2
503768 + 0 properly paired (3.23%:-nan%)
2938970 + 0 with itself and mate mapped
12681671 + 0 singletons (81.19%:-nan%)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

So this is Hiseq 100X100 PE run on mouse samples. I QC'ed by fastq using quality trimmer (>=30) and masker (>=20) and they look fairly good. I estimated the inner distance of mate paris to be 250+-200, based on a Bowtie alignment. All other parameters were kept as default.

Can anyone tell me why they pair so badly? Could it be adaptor contamination?

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05-22-2013, 02:00 PM	#1
Felix.Lee Junior Member Location: Iowa Join Date: May 2013 Posts: 1	TopHat extremely low paired mapping rate. PLS HELP! Hey guys, I have some problems with my paried-end RNA seq analysis on Galaxy. As you can see in the bam flagstat output, my tophat alignment rate is very low. 15620641 + 0 in total (QC-passed reads + QC-failed reads) 0 + 0 duplicates 15620641 + 0 mapped (100.00%:-nan%) 15620641 + 0 paired in sequencing 7109757 + 0 read1 8510884 + 0 read2 503768 + 0 properly paired (3.23%:-nan%) 2938970 + 0 with itself and mate mapped 12681671 + 0 singletons (81.19%:-nan%) 0 + 0 with mate mapped to a different chr 0 + 0 with mate mapped to a different chr (mapQ>=5) So this is Hiseq 100X100 PE run on mouse samples. I QC'ed by fastq using quality trimmer (>=30) and masker (>=20) and they look fairly good. I estimated the inner distance of mate paris to be 250+-200, based on a Bowtie alignment. All other parameters were kept as default. Can anyone tell me why they pair so badly? Could it be adaptor contamination?