[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

[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!

[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.

[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?

[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]

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.

[cibercop666]

Very very much thanks

[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!

[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.

[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]

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?

[frc1230]

As I understand it, the single-stranded sequences in the Y-adapter must match the sequences immobilized on the chip. One must be identical to one of the chip sequences, and the other must be the reverse complement of the other. I suppose in principle you could switch around which pairs with which. I can't see any advantage to doing this, though, and there could well be problems since this system is very specific (amazing to me that it works at all).

[Akira]

During PCR amplification, the primer2.0 will first bind to P7 region, synthesize the 1st PCR product, then only primer1.0 will be able to bind to its complement at P5 region, am I right?

What if I want the P7 region to be synthesized later, after the 1st strand?

Just out of curiosity, if the Y-adapter now is the reverse complement of the original single-stranded sequence, any chances it will form hairpin or any undesirable product, if now the Y-adapter is stable, the reverse complement of it will be stable as well, no?

Sorry for the poor English btw.

[frc1230]

I think in principle you are right, as far as I know. I can't personally see any reason why it would be advantageous to switch the order of synthesis of the two arms, and without a compelling reason I would not mess with the system.

[aleferna]

Hi it seems they changed the sequence of the MiSeq adapters in the new Nextera Kit, anybody knows what is the sequence of the indexing adapters in the new Nextera kits?