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Old 08-17-2011, 09:22 AM   #1
whitestein
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Location: Saskatchewan, Canada

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Default Library size selection for bacterial RNAseq

Hello,
I've been reading the forum and am confused about what size of library to select for RNAseq. We want to capture small RNAs (and all mRNA) but don't want to select too short of fragments as to miss the rest of the transcriptome information and/or have excessive sequencing into the adapter region.
The sequencing provider we've been talking to recommends Illumina 75 bp paired-end reads for RNAseq. From this, I assume that a library size of 200 bp - 400 bp would be optimal. This would mean a fragment size of ~70 bp - 270bp.
Does this sound ok?
Any advice would be helpful.
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Old 08-19-2011, 04:12 AM   #2
pmiguel
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Location: Purdue University, West Lafayette, Indiana

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Hi whitestein,
Various issues at play here. But I would say the only major issue with your plan would be that the shorter (70 bp) fragments will probably amplify preferentially during enrichment PCR.
Is this a de novo transcriptome or do you have reference to map to?
--
Phillip
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Old 08-19-2011, 06:36 AM   #3
whitestein
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Thanks for the reply Philip.
Yes, we have a reference genome to compare to.
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