[NextGenSeq]

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

[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.

[seqfan05]

Do pages 17 - 20 of this help?

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

[seqgirl123]

[post deleted]

[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!

[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.

[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]

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]

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 again, sci_guy. Makes sense.

All the best!

[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]

Hi, frc1230! Your schematic is great. Thanks for the contribution.

-Nick

[sebbaba]

Homemade TruSeq adapters

Hi all, for various reason I am generating homemade TruSeq adapters. After annealling the universal and index oligos, they run at ~65bp on a 2.5% gel as I expected. However, Illumina TruSeq adapters from the kit, run as a positive control, were at ~100bp. Anyone have any experience of why this is (given they are supposed to be ~65bp)? Is it anything to do with the Y structure and does this, presumably, mean that my homemade adapters have not annealed properly? If anything I would have thought the Y-structure would have meant they run smaller than 60bp, not larger.

Thanks
nb. To anneal I mixed eqimolar amounts of index and univeral oligos in 50mM NaCL, incubated at 95C for 5 min and allowed to cool to RT in the heat block (2hr). The oligos are modifed 5'phosphate and 3'phosphorothioate respectively.

[kulukulas]

frc1230 -> thanks for the nice schema!
for improving next version: it might be misleading when you write qPCR (from Quail et al) primers below multiplexing sequences, because they will work with only some of the indexes (last base of primer is first base of index)
otherwise very nice
best,
Lukasz

[frc1230]

TruSeq sequence manipulations for library preparation

Kulukulas, thanks for the correction on the oligos.

The attached document contains my best guess as to how the new TruSeq kit works: by a similar principle as in my previous schematic, but there's one critical difference. By the time adapter ligation is complete, the material is cluster-formation-ready. The PCR amplification is solely to amplify the material, it does not add any sequences (unlike in the previous procedure, where the PCR amplification added the 'right-hand' sequence required for cluster formation). (Illumina Tech support was willing to confirm this statement, but they do not provide the sequences of the PCR oligos. It is nevertheless reasonably clear what those sequences must be like).