I have created cDNA libraries from Drosophila melanogaster mRNA using the KAPA mRNA HyperPrep kit. The size distribution on the Bioanalyzer looks good, as I was expecting a mean of ~320 bp. I attached a screenshot of an example trace for one of my samples: My question concerns the two peaks very close to the upper marker (left-hand side): Are these adapter dimers (probably not, given their small size), or left-over primers? If not, what could they be? And, would this pose a problem for sequencing, given how much of them is present compared to the cDNA fragments? Thanks for any help!
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Role/Importance of adapter contamination in cDNA libraries
Last edited by patkrat; 08-12-2017, 08:03 AM. -
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Originally posted by nucacidhunter View Post1x bead clean up should have removed any fragment below 100 nt. I would suggest to rerun the Bioanalyzer to rule out possibility of issues with Chip run and contact KAPA tech support for their explanation.
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If you are running these on a non-patterned-flowcell Illumina sequencer, ignore the primer peaks. If you are running them on a patterned-flowcell Illumina sequencer (HiSeq 3000/4000/X and NovaSeq) then do an extra ampure (or whatever small fragment clean-up you have available to you.)
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Phillip
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Originally posted by pmiguel View PostIf you are running these on a non-patterned-flowcell Illumina sequencer, ignore the primer peaks. If you are running them on a patterned-flowcell Illumina sequencer (HiSeq 3000/4000/X and NovaSeq) then do an extra ampure (or whatever small fragment clean-up you have available to you.)
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Phillip
Can you explain your reasoning behind the difference between patterned and non-patterned flowcells?
I can see how primers that contain an index sequence might lead to more index hopping on patterned flowcells, but what would be the issue with these primers that don’t contain an index sequence?
I have noticed these primer peaks with Kapa kits before since they use a high concentration of PCR primers to prevent exhaustion, but I have never sequenced the libraries on a patterned flowcell. Now I’m curious to see if this is going to be an issue on the NovaSeq.
Thank You
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Hi dylanfofylan,
Have a look at this video: https://www.youtube.com/watch?v=pfZp5Vgsbw0
Please note how the ends of the library molecules are double-stranded when settling onto the flowcell. Any PCR primers will compete with the oligos present in the reagents - and your PCR primers will not be blocked from extension.
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Originally posted by dylanfofylan View PostHello Phillip,
Can you explain your reasoning behind the difference between patterned and non-patterned flowcells?
I can see how primers that contain an index sequence might lead to more index hopping on patterned flowcells, but what would be the issue with these primers that don’t contain an index sequence?
I have noticed these primer peaks with Kapa kits before since they use a high concentration of PCR primers to prevent exhaustion, but I have never sequenced the libraries on a patterned flowcell. Now I’m curious to see if this is going to be an issue on the NovaSeq.
Thank You
This is a good point. This article explains the issue well: http://www.molecularecologist.com/20...as-hiseq-4000/
The main problem, I gather, is that left-over primers are not washed away in patterned flow cells, and can then 'hijack' the DNA polymerase by functioning as a template for the extension of library fragments. However, I have three questions:
1. Is the main problem arising from this situation that primers containing the index can generate new library fragments with wrong indices, thereby resulting in misassignment of gene expression to samples when demultiplexing?
2. If so, primers that do not contain the index should not be a problem, correct?
3. If one uses libraries made with primers not containing the index (like KAPA primers), and one has primer carry-over as shown in my Bioanalyzer trace above, is there any disadvantage of using a patterned vs. a non-patterned flow cell? I can imaging that, even if index switching is not an issue in this case, because left-over primers are not washed away from the flow cell before clustering, they may sit around in the wells an reduce coverage?
Thanks,
PatrickLast edited by patkrat; 12-22-2017, 04:55 AM.
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