Illumina Sequencing Primer questions

[stighe]

Dear HiSeqers

Can anyone confirm the sequence of the SE v3 sequencing primer. Best I can find is 5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT 3', but unsure if that is for V2 or V3 sequencing kits...or if it even has changed.

Scott

[kmcarr]

Dear HiSeqers

Can anyone confirm the sequence of the SE v3 sequencing primer. Best I can find is 5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT 3', but unsure if that is for V2 or V3 sequencing kits...or if it even has changed.

Scott

Yep, that's it. It matches the Universal (non-indexed) adapter which is the same across TruSeq DNA & RNA kits v1, v2 & LT (formerly known as v3).

[rohan_dandage]

Hello,
I have a related query. I am set out to do an amplicon sequencing on miseq. Going through the library architectures (Illumina TruSeq DNA Adapters De-Mystified Illumina - all flavors) and adapter sequences (Illumina Customer Sequence Letter (August 12, 2014)), I have designed a strategy involving 3 simple PCR steps which skips all of recommended-kit-based-library preparation. Please have a look.

where,
P5: part of adapter that attaches to flowcell 29 nt :AATGATACGGCGACCACCGAGATCTACAC
P7: part of adapter that attaches to flowcell 24 nt :CAAGCAGAAGACGGCATACGAGAT
Read 1 : sequencing primer 33nt :ACACTCTTTCCCTACACGACGCTCTTCCGATCT
Read 2: sequencing primer 33nt :GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC
i5 :barcode 8nt
i7 : barcode 8nt

PCR1: For primer:ACACTCTTTCCCTACACGACGCTCTTCCGATCT<template for 10bp>
PCR1: Rev primer:GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC<template rev 10bp>
PCR2: For primer:GATCTACAC<i5 8nt>ACACTCTTT
PCR2: Rev primer:ATACGAGAT<i7 8nt>GTGACTGGA
PCR3: For primer:P5
PCR3: Rev primer:P7

For plexing 48 samples I would require only 24 (2+12(i5) +8(i7) +2) primers, a standard high fidelity pol and gel purification. I am very skeptical about mainly because of posts mentioning that illumina adapters are methylated so custom primers dont work and also the fact that if the process was so simple, the expensive kits may not be existing!
If anybody has any idea whether this kind of tinkering works or not, please let me know. As a test I will be doping a single such library next week. I would let know the result. Thanks.

[cosmarium]

Rohan,
How did your experiment go? DO you have comments on this that may help in the design. I am following a similar approach with two PCRs.
Best,
Carolina