Strange peaks in TruSeq RNA library

TonyBrooks · 07-06-2011, 06:48 AM

Hi all
I've run these TruSeq RNA libraries I received on the Bioanalyser HS Kit and they appear to be have strange peaks, almost as if there was a ladder contamination (although the peaks don't correlate with the HS ladder sizes). I've run these samples twice now, preparing fresh gel-dye and using a new tube of marker, but I get the same traces.
Has anyone got any explanations?

pmiguel · 07-06-2011, 08:57 AM

Hi Tony,
Just a guess, but:
(1) A MW ladder, as you mention as a possibility, was somehow introduced at some point.

(2) It could be highly expressed transcripts in your sample. They might not be visible in the original total RNA QC because the

(3) Horror of horrors, the TruSeq kit includes some crazy control fragments that are supposed to help you trouble shoot/QC your libraries down stream. I would as soon jab a pippeter in my eye as add extraneous DNA to samples during library construction. But it is part of the standard TruSeq protocol...

Here are their sizes according to the "Illumina Adapter Sequence Letter" you can request from your Illumina FAS:

CTE2 - 150bp
CTE2 - 250bp
CTE2 - 350bp
CTE2 - 450bp
CTE2 - 550bp
CTE2 - 650bp
CTE2 - 750bp
CTE2 - 850bp
CTE1 - 123bp
CTE1 - 223bp
CTE1 - 323bp
CTE1 - 423bp
CTE1 - 523bp
CTE1 - 623bp
CTE1 - 723bp
CTE1 - 823bp
CTA - 150bp
CTA - 250bp
CTA - 350bp
CTA - 450bp
CTA - 550bp
CTA - 650bp
CTA - 750bp
CTA - 850bp
CTL - 150bp
CTL - 250bp
CTL - 350bp
CTL - 450bp
CTL - 550bp
CTL - 650bp
CTL - 750bp
CTL - 850bp

Then, I guess, there would be adapter ligated on to some/all of them? The universal strand is 58 nt and the index strand is 63. After enrichment PCR (if any) the frags would be 121 bp longer?

That would give you these size frags:

244
271
344
371
444
471
544
571
644
671
744
771
844
871
944
971

Some of those might explain some of your extra peaks. The 128 bp and 3109 bp peaks do not seem to fit that hypothesis though. 128 is probably an adapter dimer, though. Maybe a concatamer of some of the control frags?

--
Phillip

bruce01 · 07-06-2011, 09:03 AM

Could be from the Ampure beads remaining in solution? I tend to leave the plate on the magnetic stand for a few minutes more than in the manual (7 vs. 5 minutes). Also check pipet tip carefully to see if any beads remain. Can be hard to take supernatant without taking up some beads. Also are you seeing this in all libraries?

By the way are you doing the standard PCR enrichment (all 15 cycles)? Not seeing the ubiquitous 'bump' ~600-2000bp, wondering why.

josdegraaf · 01-18-2012, 07:54 AM

any updates on this? we started to see something similar..

ETHANol · 01-18-2012, 08:36 AM

Quote:

Originally Posted by pmiguel

I would as soon jab a pippeter in my eye as add extraneous DNA to samples during library construction. But it is part of the standard TruSeq protocol...

ha ha.

Totally agree. Was trying to explain to a student collaborator why we were leaving out the 'in-line controls'. He didn't like my first answer so I came back with 'because it's the stupidest #$%@@$ idea I've ever heard of'.

Anyway, agree my guess would be too much of the in-line controls were added.

victorsor · 08-26-2012, 03:24 AM

We saw the same peaks but a very lower extent in some Truseq DNA libraries. Sequencing of this libraries confirmed that those peaks are truseq controls. After that, we never use again controls.

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07-06-2011, 08:57 AM	#2
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Hi Tony, Just a guess, but: (1) A MW ladder, as you mention as a possibility, was somehow introduced at some point. (2) It could be highly expressed transcripts in your sample. They might not be visible in the original total RNA QC because the (3) Horror of horrors, the TruSeq kit includes some crazy control fragments that are supposed to help you trouble shoot/QC your libraries down stream. I would as soon jab a pippeter in my eye as add extraneous DNA to samples during library construction. But it is part of the standard TruSeq protocol... Here are their sizes according to the "Illumina Adapter Sequence Letter" you can request from your Illumina FAS: CTE2 - 150bp CTE2 - 250bp CTE2 - 350bp CTE2 - 450bp CTE2 - 550bp CTE2 - 650bp CTE2 - 750bp CTE2 - 850bp CTE1 - 123bp CTE1 - 223bp CTE1 - 323bp CTE1 - 423bp CTE1 - 523bp CTE1 - 623bp CTE1 - 723bp CTE1 - 823bp CTA - 150bp CTA - 250bp CTA - 350bp CTA - 450bp CTA - 550bp CTA - 650bp CTA - 750bp CTA - 850bp CTL - 150bp CTL - 250bp CTL - 350bp CTL - 450bp CTL - 550bp CTL - 650bp CTL - 750bp CTL - 850bp Then, I guess, there would be adapter ligated on to some/all of them? The universal strand is 58 nt and the index strand is 63. After enrichment PCR (if any) the frags would be 121 bp longer? That would give you these size frags: 244 271 344 371 444 471 544 571 644 671 744 771 844 871 944 971 Some of those might explain some of your extra peaks. The 128 bp and 3109 bp peaks do not seem to fit that hypothesis though. 128 is probably an adapter dimer, though. Maybe a concatamer of some of the control frags? -- Phillip

07-06-2011, 09:03 AM	#3
bruce01 Senior Member Location: . Join Date: Mar 2011 Posts: 157	Could be from the Ampure beads remaining in solution? I tend to leave the plate on the magnetic stand for a few minutes more than in the manual (7 vs. 5 minutes). Also check pipet tip carefully to see if any beads remain. Can be hard to take supernatant without taking up some beads. Also are you seeing this in all libraries? By the way are you doing the standard PCR enrichment (all 15 cycles)? Not seeing the ubiquitous 'bump' ~600-2000bp, wondering why.

01-18-2012, 07:54 AM	#4
josdegraaf Member Location: Germany Join Date: Mar 2010 Posts: 33	any updates on this? we started to see something similar..

08-26-2012, 03:24 AM	#6
victorsor Member Location: Madrid Join Date: Dec 2011 Posts: 13	We saw the same peaks but a very lower extent in some Truseq DNA libraries. Sequencing of this libraries confirmed that those peaks are truseq controls. After that, we never use again controls.