Truseq Custom Amplicon kit overhang sequences

[mjb_2902]

I have been searching for for the overhang sequence on the i7 and i5 indices of the Truseq Custom amplicon Index kit, but couldn't find them..
We have designed about 10 primer pairs (microbiology, fungal and bacterial) and we want to link these primers, through two step pcr, to the indices of the Truseq amplicon kit (which is part of the Cancer Panel Kit from Illumina). Illumina won’t provide them since they are classified/company secret..

So far I only found information about adapters of Nextera and Truseq DNA kits.

Any help could be usefull.

[niroshan]

You should be able to find the sequences in this Document:

https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/experiment-design/illumina-adapter-sequences_1000000002694-00.pdf

[mjb_2902]

Thank you, This document only shows the index sequences (page 14) NOT the pcr primer sequences.

[SylvainL]

Do you already have a library obtained with these PCR primers? If yes, take an aliquot and make a Topo-cloning and then sequence the plasmids with M13 and T7 primers to get the sequences of the Illumina PCR primers...

[nucacidhunter]

That's a good idea and worth trying but I think it may not work due to presence of 5' blocked oligos used for amplification.

[SylvainL]

Are you sure they are 5' blocked? What would be the point to block on 5' for a PCR? I never prepared library, maybe there is a step between the PCR and the colony generation? Would be interested to have more details...

[nucacidhunter]

It is just a guess, that is why I think it is worth trying. P5 primer must have 5' biotin or other moity which is used for binding to normalization beads. Normalization beads have a limited number of strepdavidin or other molecules which interact with biotin and are saturated with library fragments resulting in equimolar normalization.

[Jessica_L]

I see what you mean. On the Illumina website, their FAQ points to the adapter sequence letter, but it only contains the eight bp sequences of the indices, not the adapter sequence itself.

They do explicitly say on the website that the adapters are not the same as the Nextera adapters, but at the very least, we know that the adapters have to work with the existing primer cocktails in the MiSeq cartridges, which contain a mix of TruSeq and Nextera primers, right? I'm seeing a lot of similarity between Nextera and RNA primers, and if Illumina claims those aren't the ones being used in the custom amplicon kit, maybe they're more similar to the standard TruSeq adapters (page 15/16 of the sequence letter).

My other thought would be to go looking for adapter sequence from other TruSeq Custom Amplicon reads from other runs, if you have any. Adapter trimming isn't a usual option for your sample sheets in Illumina Experiment Manager, so in theory there should be some adapter sequence in at least some of the reads, assuming amplicon length is small enough for the read to get all the way across the amplicon.

[Brian Bushnell]

It is very difficult to get a straight answer from Illumina. Anything you can derive from actually sequencing the molecules in their kits will probably be more useful than what they tell you on the phone.

[SylvainL]

For people who are interested, try "illumina confidential letter" in google... you will find a 30 pages pdf with all the sequences (adapters included)

s.

[Jessica_L]

For people who are interested, try "illumina confidential letter" in google... you will find a 30 pages pdf with all the sequences (adapters included)

s.

The pdf is actually an older version of the same document that others have mentioned in this thread. It has a section for the TSCA indices, but still doesn't contain the full adapter sequence.