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NGS sequencing and capture probes

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#1 genetics3



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Posted 11 August 2019 - 12:06 AM


I have two questions for you:

1.I don't understand,when a library is denatured before loading in the ngs instrument, I'll have the two complementary strands separately; so they will form two different clusters, the system recognizes them as duplicates because they come from the same molecule and give the same informations?Or if not how the system could recognize that they come from the same molecule but they are one the opposite strand of the other and so not duplicates? 


2.When I do capture enrichment with probes, usually are captured both strands of DNA, correct? So to capture both strands  I must have a pool of double strands probes? And in this case when I hybridize double strand probes with my double strand DNA, a fraction of this probes do self annealing?

Instead what about RNA probes that are single strand, how they can capture both dna strands? I have to create a probe for plus strand and one for minus strand? And they don't do self annealing? I Know that RNA-RNA hybrids are more stable that DNA-DNA hybrids...

And I read that RNA-DNA hybrids are more stable that DNA-DNA hybrids, so some companies prefer to use these type of probes. But why RNA-DNA are more stable?


Thank you very much in advance!

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