[kmcarr]

It seems clear from the photos that the NextSeq 500 has 4 lanes per flow cell, but i have been unable to discern from any of the published info thus far if each lane is individually loadable. Does anybody know?

[GenoMax]

Since the fluidics slide says that there are no tubes it seems likely that only one pool/sample can be run on the flowcell at one time.

Based on this document the libraries are loaded into the reagent cartridge like MiSeq: http://supportres.illumina.com/docum...15048776-a.pdf

[austinso]

I was initially dismissive, but the way they implemented the ordered clustering is really clever as is the idea of coding bases in binary, though I wonder like Phillip how one distinguishes between a failed incorporation of G and a bona fide event...?

[kmcarr]

Since the fluidics slide says that there are no tubes it seems likely that only one pool/sample can be run on the flowcell at one time.

Based on this document the libraries are loaded into the reagent cartridge like MiSeq: http://supportres.illumina.com/docum...15048776-a.pdf

Thanks for the pointer Geno. It is very clear from the User Guide that there is one, single port for sample loading so the is no dividing up the capacity of the flow cell. Seems like an unnecessary limitation to the utility of the platform.

[ymc]

Thanks for the pointer Geno. It is very clear from the User Guide that there is one, single port for sample loading so the is no dividing up the capacity of the flow cell. Seems like an unnecessary limitation to the utility of the platform.

Does that mean I can't load RNA to two lanes and exome to the other two in NextSeq 500?

[TonyBrooks]

Does that mean I can't load RNA to two lanes and exome to the other two in NextSeq 500?

You'd probably need to mix samples via indexing. I'm guessing as long as insert sizes are similar you should be OK but we know that some prep methods produce higher CD than others, so equimolar pooling could be more challenging.

[ymc]

You'd probably need to mix samples via indexing. I'm guessing as long as insert sizes are similar you should be OK but we know that some prep methods produce higher CD than others, so equimolar pooling could be more challenging.

Can it have a split loading kit like HiSeq 2500?

[TonyBrooks]

Can it have a split loading kit like HiSeq 2500?

Pretty sure it can't. It's very similar to the MiSeq in that there is one well on the cartridge in which you can load your denatured library. No option to cluster off instrument.

[TonyBrooks]

I've also noticed you need to load 0.06% bleach into the cartridge.
I guess there is an aggressive wash to remove the carry over seen in the MiSeq.

[ymc]

Pretty sure it can't. It's very similar to the MiSeq in that there is one well on the cartridge in which you can load your denatured library. No option to cluster off instrument.

Thanks for your reply.

What an unfortunate design choice! I hope they fix it in the next iteration

[pmiguel]

Illumina controls the clustering adapters and sequencing primers. It should be trivial to ensure no Gs at the end of the cluster.

You mean by backing away the sequencing primer from the insert? Sequencing the adapter for one or more bases?

I don't think that is a likely scenario.

--
Phillip

[GW_OK]

Yeah, that'd mean an entirely new, machine-specific library kit and backwards compatibility issues.

Is this not just a little bit nit-picky, though? Think of a genome. Think of an amplicon. How many times would arise wherein the first X bases were a G-homopolymer? Theoretically it could happen, certainly, but practically, I'm thinking not so much.

Anyone have any concrete examples of G-heavy read starts?

[GenoMax]

From the NextSeq 500 system user guide from support site.

For any index sequences, RTA2.0 requires at least one base other than G in the first two cycles (i.e. i7 -705 index is not compatible)

[jwfoley]

Here are some prices I've heard:
FC-404-1002 | NextSeq™ 500 High Output Kit (150 cycles)
List Price (USD):$2,500.00

FC-404-1004 | NextSeq™ 500 High Output Kit (300 cycles)
List Price (USD):$4,000.00

FC-404-1005 | NextSeq™ 500 High Output Kit (75 cycles)
List Price (USD):$1,300.00

Based on this information I've worked out that the cost per library on the NextSeq is almost exactly the same as the cost on the Ion Proton with the upcoming PII chip, which I assume it was intended to compete with. https://docs.google.com/spreadsheet/...k8wczNWN0kxeFE

Please let me know if you have more information than I do.

[pmiguel]

Yeah, that'd mean an entirely new, machine-specific library kit and backwards compatibility issues.

Is this not just a little bit nit-picky, though? Think of a genome. Think of an amplicon. How many times would arise wherein the first X bases were a G-homopolymer? Theoretically it could happen, certainly, but practically, I'm thinking not so much.

Anyone have any concrete examples of G-heavy read starts?

Well the stranded TruSeq RNA prep libraries tend to be quite G-rich in their early bases.

Call me cynical, but I just know this is going to cause bias unless Illumina has substantially altered its cluster calling software. I am guessing anything with 3 or more G's in its read1 first 5 bases is going to be "lost" by the software as a cluster a large percentage of the time.

Not that Illumina will have any trouble competing with the Proton.

Also, after a year or so Illumina will probably fix the issue. That is my prediction. Feel free to ridicule me if it didn't go down that way 1 year from now.

--
Phillip