I am planning to pursue a small RNA sequencing project, however, I am a bit under confident in choosing the correct read length for a illumina hiseq based small RNA sequencing. The options that I have are 50bp or 75bp, with SE or PE choices. Kindly help, and if possible provide reference to papers where people have used your suggested choice of sequencing length.
I would suggest please go for 50nt Sequencing on a HiSeq 2000 it can be Single End reads as that would be sufficient . If you generate 15-20 million reads it will be good data. Yes, all your small RNA's will be between 17-28 nt (miRNA, siRNA, piRNA, snoRNA and etc).
Both 50bp and 75 bp will work as the small rna length would be just 30 nt. SE is always the choice for small RNA sequencing. I would worry more about the coverage. You need good number of copies per small RNA for downstream analysis and prediction.
You can use 50 or 75 bp SE for small RNA. The size of small RNA is around 18-22 bp.
As they usually express at lower levels than the other transcripts, try to go as deep as you can in your sequencing (> 10 million reads, if it is possible for you financially). I think both 50bp and 75bps would work for you.
I would suggest please go for 50nt Sequencing on a HiSeq 2000 it can be Single End reads as that would be sufficient . If you generate 15-20 million reads it will be good data. Yes, all your small RNA's will be between 17-28 nt (miRNA, siRNA, piRNA, snoRNA and etc).
Thank you all for your kind assistance.
Dear Gaurab,
Thank you for the clarity provided. I was however wondering - 15-20M reads for small RNAs - isn't it a bit too much? Most of the researches that I have come across suggest that for understanding differential expression 4-6M reads are ok, and for discovery - 8-10M reads are fine. Kindly provide your views.
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