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-   -   Help understanding directional libraries in bisulfite sequencing (http://seqanswers.com/forums/showthread.php?t=89043)

emham 04-27-2019 02:53 PM

Help understanding directional libraries in bisulfite sequencing
 
Hi all, I know there are some other threads about this and I have read them, but for some reason I'm still struggling to understand the molecular biology behind directional vs. non-directional libraries. I hoping some of you can help me hash this out...

OT=original top strand
OB=original bottom strand
CTOT=complement to original top
CTOB=complement to original bottom

I know I have directional bisulfite libraries but I'm not sure why, and I have been trying to sketch out all the molecules in the process to figure out why the CTOT and CTOB strands are not read. Here's the process: I ligate fragmented dsDNA with methylated Y adapters, then bisulfite convert, then graft indices on with PCR which also adds P5 and P7 sequences.

Let's call the reverse complements of P5 and P7 P5rc and P7rc, respectively.

By my drawings, at this point the OT and OB strands look like this:
5' P5-insert-P7rc 3'

and the CTOT and CTOB strands look like this:
5' P7-insert-P5rc 3'


If the flow cell oligo lawn has both P7 and P5 oligos bound at their 5' ends to the glass, when you flow the ssDNA over the lawn then shouldn't all the molecules with P5rc or P7rc at their 3' ends (all the strands!) hyb to the flow cell and do bridge PCR?

Then, once the P5s are cleaved, the P7 read (read1) should matched the OT or OB strand, but then after P5 is re-synthesized, its read would look like CTOT or CTOB!

I'm either wrong about what the molecules look like after PCR, wrong about what's on the flow cell, missing something, or just incapable of grasping this! Any help appreciated :)

emham 04-27-2019 03:15 PM

\_(ツ)_/
 
or is it just that READ1 is always OT/OB so that dictates the orientation for the entire read pair even though READ2 is the reverse complement of that?!


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