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Old 08-09-2017, 09:19 PM   #1
hyssakura
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Question Which strand of dsDNA library is the initial template on the Flow cell

The both of complement ssDNA can hybirdize to p5 or p7 oligos on flow cell.
In this case, the sense strand and the antisense strand will form two identical clusters after bridge PCR. So the duplication rate will be 50%.

But in the skematic of Illumina, only p7 oligos can extand to be the initial template before bridge PCR. It means only one strand of dsDNA can hybirdize to P7 oligos.

So Why p5 can't hybirdize one strand of dsDNA? Are p5 oligos is blocked?
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Old 04-26-2019, 12:24 AM   #2
emham
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p5 does bind and form clusters but then the p5 molecules are cleaved and p7-attached clusters are sequenced first in read1. then those molecules are copied by bridge PCR and the p5-attached molecules are sequenced in read2
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Old 04-26-2019, 09:54 AM   #3
luc
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Quote:
Originally Posted by hyssakura View Post
It means only one strand of dsDNA can hybirdize to P7 oligos.

So Why p5 can't hybirdize one strand of dsDNA? Are p5 oligos is blocked?
Every library molecule and every strand has both the P5 and P7 sequences.
Only one of the ends hybridizes initially, because of the direction/complementarity of the flowcell oligos.
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Old 04-27-2019, 01:52 PM   #4
jdk787
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Quote:
Originally Posted by luc View Post
Every library molecule and every strand has both the P5 and P7 sequences.
This true of libraries with Y shaped adapters prior to PCR, but after PCR won't there also be strands that contain the complements to the original adapter sequences? So for a double stranded library molecule after PCR, one strand will have the P5 binding site and one will have the P7 binding site.

@hyssakura I think this blog explains the process pretty well
https://kscbioinformatics.wordpress....are-sequenced/
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