[Akira]

Many thanks for the reply.

A random thought: Is it possible to make a Y-adapter, using the reverse complement sequence of the fork? Instead of 5' end of the adapter start with P5 region, change it to the P7 region (reverse complement or whatever, but will ensure the end product is the same).

Anyone tried this?

[protist]

Hi. Is it the old PE adapters supplied in Paired End Sample Prep Kit contains a mixture of 2 types of double stranded adapters?

While the new TruSeq DNA sample prep kit only comes with one Y-adapter?

If yes for the 1st question, does it mean there is a possibility that the fragments will probably ligated with 2 same adapter at both ends, and also 2 different adapters at both ends, and only fragments with both different adapters are qualified for cluster generation?

Thank you.

1st Q - old adapters
No it is the same Y shaped adapter: the P7 has additional sequence to incorporate 2nd read sequencing primer and annealing portion for flowcell so that clustering for the second read can be performed. Also the amplification primers are different for PE libraries to reflect the changes in the P7 sequence. I have attached a doc I give to students/interested parties to illustrate the adapter structure.

2nd Q - Truseq
TruSeq adapter is a complete adapter ie it technically does not require any amplification step as all the sequences required to anneal to the flowcell are included in the adapter - the old adapters absolutely required amplification otherwise the portion that anneals to the flowcell would not be present in the library.

[Akira]

Hi. Is it the old PE adapters supplied in Paired End Sample Prep Kit contains a mixture of 2 types of double stranded adapters?

While the new TruSeq DNA sample prep kit only comes with one Y-adapter?

If yes for the 1st question, does it mean there is a possibility that the fragments will probably ligated with 2 same adapter at both ends, and also 2 different adapters at both ends, and only fragments with both different adapters are qualified for cluster generation?

Thank you.

[ehui]

Hi,
Illumina company has updated the small RNA cloning kit in 2010, known as TruSeq™ Small RNA*Sample*Preparation kit, so I wonder if the sequences of the RNA adapters and primers have beed changed. Additionally, because the manual does not provide the sequence of each linker or primer, can you kindly provide their exact sequence, expecially the 5' RNA Adapter (RA3), 3' RNA Adapter (RA5) and index containing RNA PCR primer (anyone of 48 primers)?
Thank you very much!

[cibercop666]

Very very much thanks

[RMarsh]

Phillip,
Thanks for the reply. Sorry, my post lacked specificity; my question is more of a dinosaur at this point. I was wondering about the original PE adapter/primers listed at the beginning of this thread. You are correct about the TruSeq design; it doesn’t make sense that the primers would span the entire adapter like the original PE adapter design.

[pmiguel]

During ligation the only possible product would have A-insert-B once strand-denatured. You seem to be presuming that the TruSeq primers (from the "PPC" tube) span the entirety of the adapters.

(1) Illumina, as far as I know, refuses to disclose the sequence, or any other information about the composition of their TruSeq enrichment PCR primers. Have you somehow determined their sequence? Eg, with a limited Snake Venom Diesterase digestion followed by Mass Spec, or some other method.

(2) Illumina does specify, if asked, that the PPC primers are compatible with all TruSeq indexes. Which makes it likely that they do not span the entirety of the adapters. Otherwise PPCs would likely be index specific.

Thus it seems unlikely that the TruSeq PPC primers include sequence in the insert-proximal (complementary) region.

--
Phillip

[RMarsh]

There is only one adaptor; it has a “Y” configuration with a region of complementary sequence at the end that ligates to the genomic DNA, and two different tails that correspond to the two different primers. This design should yield two different ends after PCR amplification.

My question…Illumina’s PCR oligos extend into the region of complementary sequence which could give rise, be it with low efficiency, to products that have two A ends or two B ends. I’m not familiar with a great deal of paired end sequencing results, but how often does this cause a problem (failed sequences, or reads without paired reads)?

The TUFTS design moves the priming back to exclude the complementary region, which eliminates the possibility of products with identical ends (well at least identical ends due to the adapter/PCR design). Has anyone tried modifying the Illumina PCR oligos to remove the region of complementary sequence or is this not a problem?

[wudi0616]

Hi,
Is there a chance that the sheared genomic DNA fragment ligate with both adapter 1(or 2)
at both ends? Or it's not a problem later on.

[kkusumika]

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).

thanks a lot!! makes things so much clearer!

[HGENETIC]

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).

Thanks for posting this it's really helpful and is something I'd been trying to fathom out myself recently - H

[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).

[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

[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.

[nbogard]

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

-Nick