[frc1230]

Illumina sequence manipulations

Hello, new to NGS, I've found this site extremely helpful. For my own edification and to help others in the same fog I was in, I put together a document with all the manipulations involved in single-end and multiplexed library preparation. Information from various posts on this site was critical in being able to do this. I hope that people can check this document for errors, both in overall structure of the scheme, and in the specific oligo sequences.

[nbogard]

Thanks again, sci_guy. Makes sense.

All the best!

[sci_guy]

Could be leftover activity. But I would assume that the chaotropic salts and EtOH in the column would kill the activity.

Some degradation of the 3' end in the Y-tail is not a problem. The PCR primer will still anneal nicely. However, annealing of two blunt-ended Y adaptors yields an amplifiable product.

Have a look at this thread: http://seqanswers.com/forums/archive...hp/t-1765.html

[nbogard]

Thanks sci_guy!

Is the exonuclease activity in the ligation prep from leftover T4 DNA Pol used in the earlier end-repair reaction? If the contamination is significant, that would explain the reason for the phosphorothioate modification at the ligation end. But why the lack of the modification on the opposite adapter that also contains an exposed, single-strand 3'-end?

[sci_guy]

Hi nbogard,

Exonucleases in the ligase prep can chew off the adaptor "A" overhang. Two of these blunt-end ligated together will amplify nicely. While the probability of exonuclease activity and frequency of blunt end ligation is small, the adaptor is in great excess over DNA library and will amplify later more efficiently (due to being shorter than the library).

LNAs will increase the apparent Tm, but may hinder ligation. I'm not sure if the ligase will accommodate bulky LNA backbones. You'll have to test... anyone with experience here?

[nbogard]

Why is the phosphorothioate modification (in the adapter) at the 3'-end that is proximal to the ligation? Shouldn't that 3'-end be protected after the ligation? I don't understand why the modification is not added to the Y-end of the opposite adapter. It seems that the 3'-end of only that adapter would be vulnerable to the nuclease.

Also, do locked nucleic acids (as used in the Tufts protocol) serve the same purpose: to prevent 3' to 5' nuclease activity? Do either the phosphorothioate bond or locked nucleic acids aid in the annealing of the two adapters to each other?

Any clarification or insight would be greatly appreciated. Thanks much.

[shoegame2001]

Does anyone know the concentration of the indexed adapters in the TruSeq RNA sample prep kit? Or alternatively, what concentration of custom-made primers from IDT or elsewhere have people had success with integrating into the TruSeq protocol?

Thanks!

[seqgirl123]

[post deleted]

[seqfan05]

Do pages 17 - 20 of this help?

I can not find the page17-20, How does it describe?

[seqgirl123]

NextGenSeq, it is a trick some people apply when their DNA yield is low after PCR cleanup in library prep. They amplify with qPCR primers to increase the yield, I guess this ends up more closely representing cluster amplification in some ways, so it won't necessarily create more artifacts or bias like regular PCR amplification would, but give better tighter PCR amplification of target. I am not sure if I have all the details right though about this trick. Was hoping that anyone here has tried it can comment on their experiences.

[NextGenSeq]

No, not sure why you would do that. The more amplification you do in your library prep the more artifacts you create.

[seqgirl123]

Do you ever use the KAPA GAIIx qPCR primers for library re amplification before putting the library onto the cluster station?

[NextGenSeq]

We use the KAPA library quantification kit. It comes with everything you need and works very well.

[seqgirl123]

anyone use qPCR primers during library prep?

Does anyone use the qPCR primers sold by Applied Biosystems to amplify their PCR product? Their qPCR primer sequences they sell are identical to Illumina's flowcell oligos 'P5' and 'P7'.

Taking an example, say you have a genomic paired-end library and for whatever reason, there's low library yield after PCR cleanup. So you decide to reamplify. But instead of reamplifying with the primers again (which is the common route), does anyone use the qPCR primers instead? The reasoning behind it is that your library now contains the regions complementary to flowcell oligos, so the qPCR primers can be used for reamplification instead of the PE-primers. I am not sure how this would help though. Perhaps because the qPCR primers are same as flowcell oligos, they will amplify the material better and thus will lessen PCR bias, and give higher quality sequencing data? Has anyone ever heard of this?

On another note, I haven't heard of people using the qPCR primers in place of PE primers however, and not sure if it can be done either. I haven't checked it out myself.

[genlyai]

MW007, I've heard truseq does use new seqs for some oligos, but I'm not sure which. If anyone can answer this -- possibly we can get a start just from looking at a run that's used Truseq -- that would be great.

Another thing I didn't see on here is sequences of the barcode primers (from pre-truseq days). Can anybody help with that?