Interested in RNA-Seq splicing analysis - go for paired-end shorter or single-end longer?
2
0
Entering edit mode
4.8 years ago

If I'm about to send something off to sequencing, and my main question is about splicing, am I better off with 75 bp pair-end reads or 150 bp single end?

I understand that paired-end is better than single end, and longer is better than shorter, but if I'm weighting between the two, which should I prioritze for splicing detection, longer reads or paired end?

RNA-Seq splicing sequencing • 1.4k views
ADD COMMENT
2
Entering edit mode
4.8 years ago
Amitm ★ 2.3k

Hi, Assuming the sequencing template library is ~300bp (median), then a 75x2 PE data is effectively giving you information of 75 + 75 + the insert size of ~150 = ~300bp of the genome/transcriptome. Admittedly, in practice, the avg. insert size you get is mostly << 150bp as the template lib. size selection does give you many << 300bp templates. Having said that, as far as I undstnd, a 75X2 PE should give more "information" than a 150bp SE. Also, if the organism in concern has repetitive seq. content in the transcriptome, it helps I think to have a larger "info" content in data like a 75X2 PE or a 100X2 PE. I am not sure if a longer read size of 150bp as a SE alone would be more effective in tackling repetitive content at mapping step.

ADD COMMENT
0
Entering edit mode

This answers my question perfectly. Thank you!

ADD REPLY
0
Entering edit mode
4.8 years ago
ATpoint 85k

Definitely paired-end. There is no advantage of SE over PE and PE ensures exact knowledge of the position of the fragment ends in the genome/transcriptome. Also, some tools such as salmon can now tackle GC bias with PE information, though this is mainly for DEG analysis, not sure if one can/should use this for splicing analysis, not my field.

ADD COMMENT

Login before adding your answer.

Traffic: 651 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6