Insert Size Selection In Rnaseq
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10.9 years ago
Pfs ▴ 530

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

rna selection • 5.9k views
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4
Entering edit mode
10.9 years ago
Bioquant ▴ 160

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.

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