So I've been using Tophat (running bowtie2) for various RNA-seq mapping projects and using default Tophat settings will get more than 75% mapping on first instance, but i've came across a strange scenario i was hoping to get input on.
We got a library sequenced by strand specific RNA-seq, the sequencing centre messed up and did 100bp reads instead of 150bp reads, and agreed to re-sequence at 150bp for us. In the mean time i mapped the 100bp reads and as usual got more than 75% reads mapped with default settings. On getting the same sample re-sequenced at 150bp the mapping came back at only 38% mapped. Alarm bells started ringing that some thing was up. I thought perhaps allowing more mismatches from the Tophat default will allow more reads to map; 3 mismatches -> 41% mapped, 4 mismatches -> 44% mapped.
Has anybody came across a situation where a second sequencing of the same library has lower mappability?
We got a library sequenced by strand specific RNA-seq, the sequencing centre messed up and did 100bp reads instead of 150bp reads, and agreed to re-sequence at 150bp for us. In the mean time i mapped the 100bp reads and as usual got more than 75% reads mapped with default settings. On getting the same sample re-sequenced at 150bp the mapping came back at only 38% mapped. Alarm bells started ringing that some thing was up. I thought perhaps allowing more mismatches from the Tophat default will allow more reads to map; 3 mismatches -> 41% mapped, 4 mismatches -> 44% mapped.
Has anybody came across a situation where a second sequencing of the same library has lower mappability?
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