Insert Size Selection In Rnaseq

Question

[Pfs]

What are the guidelines to choose the insert size for a Paired-End Illumina RNASEQ library?

Answer 1

[Bioquant]

It depends on what your end goal is:

If you want to just calculate the gene expression then just sequence 50 bp long single end reads. The insert size should be small (150~200 bp) as you do not want to miss the small exons (avg. size of exon is ~ 200 bp).

If you want to find splice sites then you should go for paired end sequencing, 75 bp x 75 bp (on the Illumina platform if you go above 80 bp then your quality begins to degrade for RNAseq). You should have longer insert size(250 ~ 300 bp) as you would like to increase the number of fragments that bridge a splice junction. You should target for at least 100x coverage to get > 80% transcripts reconstructed using tools like scripture / cufflinks.