[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

[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

[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.

[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.

[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]

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]

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?

[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.

[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...?

[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

[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?

[BBoy]

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

[GW_OK]

More from the same presentation, NextSeq500:



Larger than normal flowcell. Like, the size of your hand.


2-dye chemistry.


LED light sources and cellphone cameras. Low cost, light weight, no alignment needed on install.


Fluidics module. "Valve and stagger" technology allows for chemistry and imaging at the same time.


2 different flowcells.


Price per Gbp

[GW_OK]

So I've been watching Jay Flatley's taped talk to investors from yesterday and I've taken some screenshots and notes for your enjoyment:


Better chemistry including new cluster method, better scan mix, speeding up the paired end turnaround chemistries, new custom camera that can do *bi-directional* scanning (allows for 6x speedup), higher powered lasers, new computer system (bye-bye Dell tower) and re-written software


Remember when Ion Torrent said they were going to use chip foundries to change the sequencing world? Wafer-scale flowcell generation for patterned FCs, 12 FC/wafer.


400nm feature size. Top and bottom of FC.


Example patterned FC picture.


700nm center-to-center distance for 400nm-sized features. Plenty of headspace to pack more and more in.


Seeding the current FCs is a problem b/c DNA binds randomly and you can get polyclonal clusters (Poisson distribution). Current size of clusters and camera power can discern polyclonal clusters fairly well, but with 400nm features there's no way to tell them apart.


New cluster chemistry has simultaneous seeding and amplification. Instantaneous amplification of the template as soon as it sits on a cluster site prevents two templates from seeding the same feature via competition. 80% monoclonal features vs 40% from regular FCs.


"Typical" data qualities from a HiseqX, ~120Gbp/lane. 1.8Tbp/run is conservative. 36x human genome/lane.

System is *licensed only for human WGS*, so that's how they keep you from running other stuff.

[TonyBrooks]

Alright, different instrument. Probably won't have the issues associated with the HiSeq (bubbles.)

Let's go back to cluster recognition. Looks like 5 initial G's and the cluster will not be seen at all. But, as you mention, that would be less than 0.1% of the amplicons. So the bias would be unlikely to cause issues.

How about 4G's? Do you really think the software would be confident enough of a cluster which only was visible in a single cycle out of the first 5?

How about 3G's?

This whole inferring signal from a lack of signal makes me twitchy...

--
Phillip

I guess that could be why cluster density is so low.