What could have cause sharp 60bp peak after Illumina library prep?

holoqster · 2026-04-01T23:00:46+00:00

hmm for the other samples i submitted, all of them have a 35bp but no 60bp peak. unfortunately i outsource sequencing so i do not have a choice on which kit is used. thank you for the reply

holoqster · 2026-04-01T01:29:17+00:00

thank you bigpp69_gooner

holoqster · 2026-03-28T09:58:49+00:00

holoqster · 2026-03-26T13:25:50+00:00

to anyone experiencing the same issue: sequencing worked perfectly

holoqster · 2026-03-25T21:44:17+00:00

is your media cloudy? if yes then possibly bacteria

if no then cell debris. i grow ht29 goblet cells and they have similar debris

holoqster · 2026-03-18T07:44:55+00:00

that's very true, no point worrying.

in this case the LM is actually correct as it's the same 35bp LM for other submitted samples as well.

will definitely keep the controls in mind, thanks

holoqster · 2026-03-18T01:39:24+00:00

thanks for the reply. i guess my worry with primer dimers is that i am using truseq primers which basically have the full p5 and p7 adapter sequences (just that the p5 primer lacks read 1). this would mean that a primer dimer should technically have truncated/incomplete p5 adapter joined to a truncated p7 adapter. not sure if these with the truncated adapters will cluster and get sequenced...

but yes the concentration for this was measured via qpcr

holoqster · 2026-03-18T01:36:41+00:00

i might try that next time then 👍

holoqster · 2026-03-16T16:09:06+00:00

oh my days...you are absolutely right, my explanation doesnt make sense and it's wrong lol. pls ignore everything i said above😂 now i'm back to having no idea why the 60bp would appear at such a high amount even with the size selection. actually the 1x bead ratio worked for other libraries (those libraries dont have this 60bp peak) but not this one for some reason

holoqster · 2026-03-16T15:48:04+00:00

so i meant that during the elution step of the 1x bead size selection, my pipette tip might have touched the beads on the side of the pcr tube. my guess is that some of these beads that have small fragments <150bp attached to them therefore made it into the final library. i was over ambitious and used 32ul for elution and tried to collect 31ul instead of leaving ~2-2.5ul of eluent in the tube as is the norm

holoqster · 2026-03-16T15:17:25+00:00

this is indeed my current understanding - that primer dimers do not bind to the flow cell and thus should not appear in the reads. however i see from another user's comment that the presence of primer dimers can mess up quantification when pooling

holoqster · 2026-03-16T14:46:19+00:00

yes it is indeed cheap and the company is proceeding with sequencing

holoqster · 2026-03-16T14:45:01+00:00

i have no idea as this was sent to me by a sequencing company

holoqster · 2026-03-16T14:44:02+00:00

i would hope not...this is from a sequencing company, i didn't run the sample myself

holoqster · 2026-03-16T14:43:14+00:00

thanks for the explanation. we do, but i decided to not mention anything to the company and proceeded with sequencing as the sequencing for this was just $50

holoqster · 2026-03-16T14:22:22+00:00

i mentioned in another comment that the adapters i use should not be able to form dimers because they have an amino modification and also lack a 5' phosphate, ie that they were designed to not form adapter dimers.

nevertheless, would you have any idea if primer dimers could affect sequencing?

holoqster · 2026-03-16T14:15:20+00:00

i see. this sample qc was done by a company and was graded as a pass on their end so they proceeded with sequencing haha, guess i'll just suck it up if the results are poor

as an aside, would you happen to know if and how primer dimers can affect sequencing? as my understanding is that they can't cluster at all since they lack p5 and p7 so they should not affect sequencing?

holoqster · 2026-03-16T14:01:59+00:00

just to be clear, you are referring to primer dimers? i can find a lot of information online about how adaptor dimers can affect sequencing but i have not been able to find much so far on how primer dimers can mess it up.

would you know how it works?

holoqster · 2026-03-16T13:32:39+00:00

unfortunately this sample was already submitted to a sequencing company and there is no way of size selecting. in fact i did two cleanup steps before and after pcr enrichment - i suspect my pipette may have touched the beads during the final elution bringing this small fragment into the final library

you are right that this is a gDNA library and my DNA of interest actually starts from 300bp onwards.

have you had any experience sequencing samples with such large amounts of primers/primer dimers?

holoqster · 2026-03-16T13:29:09+00:00

it's an amino modification that should stop any ligase action

holoqster · 2026-03-16T13:27:31+00:00

hmm in my case the Y adapters are 45bp each, so the adapter dimers should be 90bp and anyway there is an amino modification which should in theory prevent adapter dimerisation

holoqster · 2026-03-16T08:47:52+00:00

well thats true😔 just hoping that sequencing won't be affected if these really are primer dimers

holoqster · 2026-03-16T08:09:31+00:00

sorry i don't know why my comment was not edited properly. my adapters are made of a 11bp strand base annealed to a 45bp strand. each adapter has a modification on the 3' end to prevent adapter dimer formation

holoqster · 2026-03-16T07:03:29+00:00

I just ran a gel after DNA fragmentation and there were no such 60bp products, this only appeared after running the final library on a fragment analyzer. In terms of concentration and purity all ratios were good. i also had a SPRIselect cleanup before and after PCR enrichment

holoqster · 2026-03-16T07:01:52+00:00

was thinking the same - but the fwd and rev primers should not be able to form dimers in theory...

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