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Old 03-09-2018, 09:19 AM   #3
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Location: Cambridge

Join Date: Sep 2010
Posts: 115
Lightbulb I would go for PE reads in this case.

Assuming yours RNAseq libraries are compatible with patterned flowcells:

and you use dual unique barcodes for demultiplexing to reduce index crosstalk, I would go for 2x50bp reads.

But given you probably would like to also see novel splice junctions in addition to annotated ones one may go with longer reads - use a 200cycle or 300cycle kit in PE mode, than use FLASH or similar tool to preassemble paired reads, and this would give you both "SR"(combined) and PE(non-combined) reads from yours 300bp library.
The longer the reads, the higher the "SR"(combined) proportion would be. and the more resilient it should be to the repeats.

PS: if you can find a nextseq 2x150 bp RNASEQ experiment in the SRA, than you can generate both 1x100 and 2x50bp reads from it in silico by trimming, and than compare the results after analysis by your pipeline.

PPS: If you have 200 or 150 cycle S1 kit - run it as 2x100 or R1:100+R2:50 and generate both 100 SE and 2x50PE datasets from the same run by doing custom trimming. This should give you a very good idea which method would be the best in your case for future experiments.
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