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?
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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.pdfLast edited by GenoMax; 01-18-2014, 08:40 AM.
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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...?
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Originally posted by GenoMax View PostSince 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
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Originally posted by kmcarr View PostThanks 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.
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Originally posted by ymc View PostDoes that mean I can't load RNA to two lanes and exome to the other two in NextSeq 500?
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Originally posted by TonyBrooks View PostYou'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.
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Originally posted by TonyBrooks View PostPretty 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.
What an unfortunate design choice! I hope they fix it in the next iteration
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Originally posted by BBoy View PostIllumina controls the clustering adapters and sequencing primers. It should be trivial to ensure no Gs at the end of the cluster.
I don't think that is a likely scenario.
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Phillip
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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?
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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)Last edited by GenoMax; 01-21-2014, 07:23 AM. Reason: Removed the ref to "G"s since Philip has already posted that previously
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Originally posted by SNPsaurus View PostHere 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
Please let me know if you have more information than I do.
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Originally posted by GW_OK View PostYeah, 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?
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.
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Phillip
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