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Old 08-11-2019, 12:08 AM   #1
ChiaraGen
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Default NGS sequencing and capture probes

Hello!

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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Old 08-11-2019, 08:36 AM   #2
Bukowski
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Q1: Watch this: https://www.youtube.com/watch?v=fCd6B5HRaZ8

Q2: There is no requirement for your probes to be dsDNA. Why are you so interested in capturing both strands? They have exactly the same sequence right?

RNA-DNA is more stable "because thermodynamics". Im sure there is plenty of literature on the subject: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC342109/ And plenty more references here: https://www.quora.com/Why-is-the-RNA...NA-DNA-pairing

Last edited by Bukowski; 08-11-2019 at 08:39 AM.
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Old 08-12-2019, 10:43 AM   #3
ChiaraGen
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Thank you very much for your help!
What about Q2, I have found a paper
https://www.nature.com/articles/s41598-017-03448-8

where the author writes:

"DNA exists naturally as a double-stranded molecule, where one strand is encoded as a complementary molecule to the other strand. Based on this information redundancy, if the sequences of the two strands can be determined individually, it is possible to correct most if not all PCR and sequencing errors by calling a perfect match between the two complementary DNA strands. Such methods were termed as “duplex sequencing”. Several NGS studies have reported that, through duplex DNA sequencing approach and scoring only the SNVs with matched complementary sequences on both DNA strands, PCR errors and sequencing errors can be removed"

What do you think about it?
What about RNA probes, I read that RNA generates R-Loop when hybridizes with DNA, but this event happens always?Do you think that a RNA probes can generate a R-loop?
Thank you again!!

Last edited by ChiaraGen; 08-12-2019 at 10:54 AM.
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Old 08-12-2019, 12:08 PM   #4
Bukowski
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Duplex sequencing is obviously better, but requires the use of MIDs. Without MIDs there is no way to assign which half of the duplex you're sequencing with a standard library prep and Illumina sequencing. This is why I linked the Illumina video, this is inherent in the method.

I assumed R loops were formed with mature RNA transcripts. The RNA probe lengths in a target capture might be too short for this to take place, but that's just a guess. Don't know, not an expert.
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