3' bias

[numfar]

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?

[GenoMax]

See this previous SeqAnswers thread: http://seqanswers.com/forums/showthread.php?t=9839

[numfar]

The thread does not explain what 3' bias is.

[GenoMax]

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

The thread does not explain what 3' bias is.

[numfar]

So the mRNA has been fragmented? Is this intentionally?(if so why?)

[TonyBrooks]

Originally Posted by numfar

So the mRNA has been fragmented? Is this intentionally?(if so why?)

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)

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
Sequence