double peak in mRNA truseq kit

[ssing]

Hi all,

I have been doing some mRNA TruSeq sample prep, using the protocol *exactly* as advised by the Illumina folk (using PolyT beads to get mRNA, fragmenting for 7 minutes chemically, 15 PCR cycles), and I am getting an odd and consistent pattern where I have my desired peak ~300 bp and a second peak ~900 bp. See an exemplar trace attached.

Reading through the other posts, seems like most people think this due to overamplification... could that be my issue here? My biggest concern is (because the double peak is similar in size ratio to the rRNA size ratio) that this reflects rRNA contamination. Does anyone have experience with that?

Thanks!

[Prosuite]

The extra peak is due to SPRI bead carryover from post-PCR clean-up step. Use a
strong magnet for bead separation and pipette carefully during
elution to avoid disturbing beads. It should not affect the sequencing run.

[pmiguel]

Hi Prosuite,
That is very interesting. Where did you find that document from Agilent? I was just looking at the instrument troubleshooting guide this morning and there was no mention of SPRI/AMPure beads.

--
Phillip

[Prosuite]

It is from Agilent eSeminar "BioAnalyzer Applications for Next Gen Sequencing : Updates and Tips-20110301". Here is the link: https://agilenteseminar.webex.com/ag...114f10d5514079

[pmiguel]

Never seen this before. Interesting. Anyone buy the "ligase remaining attached to library molecule" issue presented on page 26?

--
Phillip

[ETHANol]

Delete what I wrote. I didn't read yours carefully enough. I doubt it is an over amplification issue because that peak usually is 2x the size of the correct peak.

[pmiguel]

Delete what I wrote. I didn't read yours carefully enough. I doubt it is an over amplification issue because that peak usually is 2x the size of the correct peak.

Did you check out the Prosuite attachment? Some info from Agilent claiming that SPRI beads can cause that result. (I have see it -- a peak much larger than one would expect from multimers/heterodimers/bubble amplicons, etc.)

If so had you ever seen that info? Be nice if Agilent provided that as part of their normal troubleshooting manual...

--
Phillip

[ssing]

When I contacted Illumina, they gave me three possible explanations:
1. Incomplete fragmentation (which seems unlikely, given that I was using RNA within the range of what the recommended and a long fragmentation time)
2. Over-amplification (which I also doubted because I didn't do a ridiculous number of cycles)
3. Bead carryover (which made the most sense as this was a consistent pattern)

If it is bead carryover, when I run a normal gel of the cleaned product, I shouldn't see that smear anymore. I'm going to do that tomorrow. I'll report back what I see. Here's hoping that is what it is, because otherwise, they are recommending I do a size selection for all my samples...

Thanks for all the feedback.

[ECO]

Never seen this before. Interesting. Anyone buy the "ligase remaining attached to library molecule" issue presented on page 26?

Chiming in with two things from my experience that may be relevant...not commenting on "page 26" because I can't see where you're referring to that....

1. Intact ligase has a strong protective effect against any treatments with exonuclease. Denature the ligase or strip out the Mg++ and you're good to go.

2. The bioanalyzer (particularly the high sensitivity kit) can be used as a gel-shift assay to detect bound proteins (DNA polymerases in my experience)...it wouldn't surprise me in the slightest that active ligase causes a shift on the bioanalyzer....in fact that's probably why ILMN adds "stop solution" to their ligations.

[pmiguel]

Bound ligase shifts Illumina libraries sizes?

Chiming in with two things from my experience that may be relevant...not commenting on "page 26" because I can't see where you're referring to that....

Sorry, Prosuite posted a link to the presentation in question above. You can skip to page 26 fairly easily. You do have to register so Agilent can spam you -- but I would say the information in the presentation is worth a little spam to anyone that uses the BioAnalyzer much.

The slide in question shows two electropherograms of an Illumina library after adapter ligation and PippinPrep size selection. One PippenPrepped after a proteinase K treatment, one without treatment. The peak with treatment is 368 bp. The one without is 291 bp.

Actually this addresses one of those perennial Illumina library prep questions that shows up on SeqAnswers. But I have never seen ligase binding suggested as the cause.

1. Intact ligase has a strong protective effect against any treatments with exonuclease. Denature the ligase or strip out the Mg++ and you're good to go.

2. The bioanalyzer (particularly the high sensitivity kit) can be used as a gel-shift assay to detect bound proteins (DNA polymerases in my experience)...it wouldn't surprise me in the slightest that active ligase causes a shift on the bioanalyzer....in fact that's probably why ILMN adds "stop solution" to their ligations.

Good to know.

--
Phillip

[ssing]

In fact, this does not appear to be due to beads. I am not sure what the peak is, but I am going to size select ... trivial exercise and worth ensuring good cluster generation.

[pmiguel]

So you saw the same phenomenon on your agarose gel? Any chance you could post that?

You might want to strand denature the sample (94 oC for 2-4 minutes followed by "snap cool" on ice) and run it on an RNA chip.

You can get double peak artifacts in TruSeq libraries that end up causing no real issues when clustered. Normally these peaks are closer in size to one another than the image you give above, though.

--
Phillip

[ssing]

Hi Philip,

See attached. The ladder is a 100 bp ladder (NEB).

[pmiguel]

(The upper loading dye is really distracting. Xylene cyanol?) Is this TBE EtBr? How many cycles of enrichment PCR did you do?

Anyway, yes there is a high molecular weight band. No harm in doing a size selection. Actually we nearly always size select (after pooling most or all of the indexes that will go in a lane). I doubt the high MW stuff will cause any issues, though.

Still, mysterious. Wonder if it could be the polymerase still bound to some of the molecules. (See ECO's comments above...)

--
Phillip

[ssing]

This is TAE EtBr and I did 15 cycles of enrichment PCR.

Not sure what this is, but I went ahead and size selected. I'll let you all know when I get my data...hopefully this is just a weird artifact and doesn't suggest anything went wrong with my library prep.