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What is 3' bias exactly? I understand it is something caused by the enrichment of poly(A) mRNA using oligo(dT) primer.(or is that wrong?) How does it cause the 3' bias?
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See this previous SeqAnswers thread: http://seqanswers.com/forums/showthread.php?t=9839 -
The thread does not explain what 3' bias is.Comment
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Since polyA tails are at the 3'-end of mRNA's any method that uses polyA sequence for enrichment is likely to recover fragments from that end.
This leads to a greater chance of having sequences from the 3'-end in the resulting reads as opposed to reads that represent the 5'-end of the molecules.
Originally posted by numfar View PostComment
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So the mRNA has been fragmented? Is this intentionally?(if so why?)Comment
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Next generation sequencers, specifically those used for RNA-Seq experiments, require library fragments around 3-400 bases. However, RNA needs to be of good quality (non fragmented) at the beginning for successful mRNA enrichment (or rRNA removal)Originally posted by numfar View Post
Typical RNA-Seq workflow goes a bit like this (based on Illumina TruSeq method):
Enrich mRNA (usually PolyA enrichment by Oligo dT beads x2)
Fragment mRNA to required size (usually metal ion-catalyzed base hydrolysis fragmentation)
Reverse transcribe to make double stranded cDNA
Library prep (end repair, adenylate, ligate adapters, size select, PCR enrich)
QC and Quantify library
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