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Old 01-25-2018, 07:17 AM   #1
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Location: Germany, Bochum

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Default Illumina fusion primers - Nextera or TruSeq based?

Dear all,

We have been using (old version) TruSeq fusion primers (Illumina flow cell bind, + sequencing primer bind + inline tag + amplification primer) for a while and would now like to add indexing to the P5 and P7 to pool more samples on the same run (Elbrecht Leese in the pictures). When looking into this, we discovered that:

1) We are using a wrong sequences on the P7 (Rd1 SP)
2) When looking at published fusion primer system, mostly TruSeq and Nextera are used (see pictures). But what really confuses us, it that the sequences in the nextera and truseq bases systems are very different! How can this be?

This is really confusion, because I always thought there is one standard index read set included with illumina sequencers, but the different sequences would indicate that for one system custom sequencing primers would have to be added? Am I missing something here?

We would like to have a ready to load fusion primer system, that uses indexing from illumina without the need to provide custom sequencing primers / and that works on all illumina systems (especially MiSeq and HiSeq rapid run). Any advide which (TruSeq or Nexters) to choose here?

What are the advantages of each when used as a one step fusion primer? Both seems to be used in the literature. OR to ask the question differently, what are the actual standard sequencing / indexing primers used, and do they differ between instruments?

Thanks every one = )

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Old 02-26-2018, 01:06 AM   #2
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Most (if not all) systems/kits support both versions. At least MiSeq and HiSeq rapid run do. They come with a mix of primers. So it is your choice which system to pick. Both work.
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Old 03-14-2018, 11:14 AM   #3
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Illumina reagent sets include primers mixes that work for various adapters. For instrument/reagent sets that pre-date the acquisition of Epicentre by Illumina, it is necessary to use custom primers for Nextera libraries. This includes HiSeq 2000/2500 high output chemistry. However the Nextera primers were added to HiSeq 2500 Rapid chemistry reagents and all MiSeq cassettes as mention by Vinz above.

The mix isn't all that complex, I would think:
(1) normal TruSeq,
(2) phiX -- which is the same as normal truseq on the i5 side but different on the i7 side,
(3) the old "RPI" style adapters that are still used (alas) in all small RNA library construction kits (anyone know of an exception?) and
(4) Nextera

But each instrument has its own implementation of these primer mixes and I have the sense that they are different lengths (ie, annealing temps). Most extreme is that some instruments sequence the i5 index (if any) via the flowcell oligo, but a few systems: NextSeq and HiSeq 3000/4000/X(not 100% sure) don't and they actually have an extra primer mix. The NovaSeq returned to the i5 index sequenced from the flowcell oligo paradigm--which I prefer. That way you know, no matter what, if you have a cluster, you will read some sort of i5 index sequence. Too bad that isn't the case for the i7 index. Would make some issues much, much easier to troubleshoot.

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