Hello,
I am new to this forum and I would love your opinion on some peculiar size profiles that we observe after our home-made library prep.
We digest the DNA, ligate adapters, perform 2 times 1xSPRI to remove the adapter dimers, then perform a PCR of 7-10 cycles and finally do a BluePippin size selection (in the range of 390 to 650 bps). After the size selection, we check the library using the fragment analyzer (Advanced Analytical) and often observe additional peaks outside of the selected BluePippin size range (see attach, sample 1 and 5). Reducing the number of PCR cycles seems to reduce the occurrence of these additional peaks (attach, sample 2). When we sequence the library, it turns out that the additional peak can be mapped to the genome and corresponds to genuine restriction digest fragments that are smaller than the selected BluePippin size range.
Question 1: What do you think may cause the small peak of short restriction digest fragments that are co-eluted in another size-range in the BluePippin?
Strolling through this forum, I encountered an interesting theory about single stranded fragments or smaller fragments or adapter dimers co-migrating anonymously with bigger fragments. Since this kind of fits most with our library size profiles, we decided to perform a QC of the library via denaturation and analysis on an RNA chip (as suggested in http://seqanswers.com/forums/showthread.php?t=12523). We analysed both non-denatured samples (red) as well as the denatured sample (2 min @ 95 and snap-cool on ice, blue) on an RNA Nano chip; the results are shown in attach.
Question 2: We observed that the Bioanalyzer with the RNA Nano chip does not detect the additional smaller peaks which we see on the fragment analyzer with the DNA chip. What could be the reason for this?
Question 3: We also observe that the denatured library gets a larger size profile than the non-denatured library, and double humps start to appear. What could be the reason for this?
Many thanks for your suggestions!
I am new to this forum and I would love your opinion on some peculiar size profiles that we observe after our home-made library prep.
We digest the DNA, ligate adapters, perform 2 times 1xSPRI to remove the adapter dimers, then perform a PCR of 7-10 cycles and finally do a BluePippin size selection (in the range of 390 to 650 bps). After the size selection, we check the library using the fragment analyzer (Advanced Analytical) and often observe additional peaks outside of the selected BluePippin size range (see attach, sample 1 and 5). Reducing the number of PCR cycles seems to reduce the occurrence of these additional peaks (attach, sample 2). When we sequence the library, it turns out that the additional peak can be mapped to the genome and corresponds to genuine restriction digest fragments that are smaller than the selected BluePippin size range.
Question 1: What do you think may cause the small peak of short restriction digest fragments that are co-eluted in another size-range in the BluePippin?
Strolling through this forum, I encountered an interesting theory about single stranded fragments or smaller fragments or adapter dimers co-migrating anonymously with bigger fragments. Since this kind of fits most with our library size profiles, we decided to perform a QC of the library via denaturation and analysis on an RNA chip (as suggested in http://seqanswers.com/forums/showthread.php?t=12523). We analysed both non-denatured samples (red) as well as the denatured sample (2 min @ 95 and snap-cool on ice, blue) on an RNA Nano chip; the results are shown in attach.
Question 2: We observed that the Bioanalyzer with the RNA Nano chip does not detect the additional smaller peaks which we see on the fragment analyzer with the DNA chip. What could be the reason for this?
Question 3: We also observe that the denatured library gets a larger size profile than the non-denatured library, and double humps start to appear. What could be the reason for this?
Many thanks for your suggestions!
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