Role/Importance of adapter contamination in cDNA libraries

[patkrat]

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!

[jdk787]

Those peaks are most likely leftover primers, which should not be a problem since they won't cluster.

[patkrat]

Those peaks are most likely leftover primers, which should not be a problem since they won't cluster.

Thanks, Josh. So, I could increase the cycle number during library amplification by 1 or 2, and see whether the peaks disappear.

[nucacidhunter]

I wonder how you have cleaned up the library. Those peaks should not remain after clean up and they can reduce cluster density/read number by blocking flow cell oligos.

[patkrat]

I wonder how you have cleaned up the library. Those peaks should not remain after clean up and they can reduce cluster density/read number by blocking flow cell oligos.

I do a 1X bead-based cleanup with KAPA Pure Beads.

[nucacidhunter]

1x 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.

[patkrat]

1x 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.

I did re-run the chip and contacted KAPA tech support. The latter suggested making sure after the library amplification to be very stringent with the 1X clean-up; i.e. remove all supernatant, and esp. all EtOH, etc. Now, I did a whole new library prep on the same samples yesterday, and made absolutely sure to follow all those steps, but still see this extra peak at ~38-40 bp. The 50 bp peak seems to be primer-dimer bleed-through that tends to happen with this protocol apparently (even visible in KAPA's tech manual..), but I don't know what these 38-40 bp peaks are. Could they be a chip artefact? I have read that the Bioanalyzer can be off by up to 5 bp at times, but even if that was the case, this wouldn't explain my situation, since the concentration of the 35 bp peak (upper marker) is 125.00 pg/ul. Or maybe I have a false understanding of how this concentration is measured..

[nucacidhunter]

If those are artifact of Bioanalyser then they should be present in the ladder as well.

[pmiguel]

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.)

--
Phillip

[dylanfofylan]

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.)

--
Phillip

Hello 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

[luc]

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.

[patkrat]

Hello 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

First of all, how do you know KAPA primers don't contain the index sequence? They didn't specify that in the TDS I purused..




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,

Patrick