[Brian Bushnell]

Illumina's current indexing strategy (up to 384 samples for Nextera) can be used on NovaSeq but using current TruSeq adapters only 8 unique combinations of index 1 and 2 is possible which is not enough for lots of applications.

It only works for applications that are not sensitive to crosstalk. Personally, I would never multiplex samples of the same genus on a NovaSeq unless all libraries had dual unique barcodes. The current system of unique pairs, in which multiple libraries share the same first barcode and multiple libraries share the same second barcode, and any pair of first and second barcodes lands a read in a specific library, is insufficient for NovaSeq. To be honest, it was insufficient for the HiSeq 2500 as well, in situations where low-frequency variants were important (or for multiplexed single-cell sequencing). But it's more insufficient now, where multiplexing is desired on a NovaSeq, due to the substantially increased crosstalk rate.

[nucacidhunter]

I agree Brian. It will be even worse if they release S4 flow cells without individual lane loading device/fix which currently is in beta testing.

[nucacidhunter]

In that case we need to have first 4-5 bases as a random sequence (for clusters ID), followed by the barcode (another 4-8 bases), than the insert...

This is really important for RNAseq & CHIPseq applications, where we want to keep the cross-talk bellow 10^-6...
Classical dual unique indexing would get us into 10^-4 - 10^-5 range.

BTW: Actually the spatial signal separation from the patterned flowcell clusters should resemble more the 454 signal, and the clusters should be much more easier to call, even if low diversity at the first few bases.

Adding 4-5 diversity base is possible but two points to consider:
1- it will be difficult to obtain good oligo annealing to form adapters and also there will unpaired oligos which would require further purification
2- Illumina is unlikely to change their established indexing strategy and also there might be some patents on inline barcoding

Unique duel indexing (every library with different index 1 and 2) should enable identification and filtering of any fragments resulting from index hopping.

Actually in patterned flow cells position of each nano-well which would have clusters is already known. I have not thought about the need for sequence diversity but it could be related to background signal.

[pmiguel]

Thanks. IDT has not released their UDI adapters that they are co-developing with Illumina. NuGEN adapters are not sold as a stand alone product and I am not sure if their adapters are specific to their kits or if they can be used with other kits as well.

You can buy the UDI 96 and/or the UDI 384 kit from IDT as a custom order. Just contact their sales. We bought the UDI 96. Made libraries (both no Amp DNA and RNAseq libraries) from them. Ran them on our NovaSeq.

The UDI 384 are 10 base indexes though. So you would have to reduce your sequence insert reads by 2 bases each to compensate...

NuGEN sales reps told me that their adapters don't use T/A tailing. So they wouldn't work with most other kits.

--
Phillip

[pmiguel]

It only works for applications that are not sensitive to crosstalk. Personally, I would never multiplex samples of the same genus on a NovaSeq unless all libraries had dual unique barcodes.

Did you see much phiX index hopping? Searching the "undetermined" fastq for index hops involving one of i7/i5 GGGGGGGG/TCGTAGTG I tentatively identified a fair number. Of course they may just be the result of signal bleed.

--
Phillip

[kcchan]

This is really important for RNAseq & CHIPseq applications, where we want to keep the cross-talk bellow 10^-6...

How did you come up with that 10^-6 figure? In an RNASeq or CHIPseq experiment that's only about 20-30 reads.

[Brian Bushnell]

Did you see much phiX index hopping? Searching the "undetermined" fastq for index hops involving one of i7/i5 GGGGGGGG/TCGTAGTG I tentatively identified a fair number. Of course they may just be the result of signal bleed.

--
Phillip

I have not looked into that yet. Actually, I don't even know if we are spiking PhiX into our Novaseq runs, but that rate is worth examining, after I find out whether there is actually any PhiX present.

[luc]

I am not sure that index hopping onto PhiX reads would be a good measure. As far as I remember the Illumina PhiX adapter sequences are quite different from TruSeq or Nextera sequences.

[pmiguel]

I am not sure that index hopping onto PhiX reads would be a good measure. As far as I remember the Illumina PhiX adapter sequences are quite different from TruSeq or Nextera sequences.

Well, not as different as you might think. Please see the attachment.

--
Phillip

[austinso]

For those with NovaSeqs, are you allowed to say how much the consumables are? Based on what people are charging for a lane, it looks like about $40K per run...

[gringer]

I was told by an Illumina rep at Queenstown Research week about a month ago that NovaSeq run cost would be similar to HiSeq, but produce about double the output.

[misterc]

List price on S4 reagent kits is about $30K. For NovaSeq sites with ≥5 NovaSeq instruments, they're able to get a discount large enough to achieve the "20% cheaper than HiSeq X" statement Francis deSouza made when they announced this platform. Something tells me this is factoring in how many samples could be run per year on NovaSeq than HiSeq X due to the throughput and runtime, so their discount is likely not more than 20% off of list. Everyone else likely gets <10% discount. HiSeq 4000 (300 cycle) kits are about $17K list price, so they're not the same price but S4 will give you about 5x the output (in about half the time).