Low mapping rate for RNAseq 2x150 trimmed data

Markovia · 08-30-2013, 07:55 PM

I just got a data set from my collaborator. 2 X 150 bps, Illumina RNAseq data for human samples. We did QC on the data and trimmed it to remove adaptors and low quality bases. Then I use tophat2 to map them to hg19 in three ways:

1. paired end mapping with default parameters;
2. single end mapping for each end separately;
3. paired end mapping with inner distance between the two ends to have a mean of 0 and sd=50;

Mapping rate for 1 and 3 are both around 45%. But mapping rate for 2 is around 75%. So most of the reads are mappable but cannot be paired even when the inner distance between the two ends can be 0 or negative (if the distance can go negative). I took a look at the BAM files for the accepted hits. It looks like read pairs with alignment

------------>>>----------------
------------<<<<----------------

can be paired. But read pairs with alignment

-------------->>>>--------------
-----------<<<<---------------

can not be paired. Does any one know why and how I can gain those unpaired alignment back? Thanks in advance!

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08-30-2013, 07:55 PM	#1
Markovia Junior Member Location: United States Join Date: Aug 2013 Posts: 1	Low mapping rate for RNAseq 2x150 trimmed data I just got a data set from my collaborator. 2 X 150 bps, Illumina RNAseq data for human samples. We did QC on the data and trimmed it to remove adaptors and low quality bases. Then I use tophat2 to map them to hg19 in three ways: 1. paired end mapping with default parameters; 2. single end mapping for each end separately; 3. paired end mapping with inner distance between the two ends to have a mean of 0 and sd=50; Mapping rate for 1 and 3 are both around 45%. But mapping rate for 2 is around 75%. So most of the reads are mappable but cannot be paired even when the inner distance between the two ends can be 0 or negative (if the distance can go negative). I took a look at the BAM files for the accepted hits. It looks like read pairs with alignment ------------>>>---------------- ------------<<<<---------------- can be paired. But read pairs with alignment -------------->>>>-------------- -----------<<<<--------------- can not be paired. Does any one know why and how I can gain those unpaired alignment back? Thanks in advance!