A heads up for all NextSeq 500 users!

[LizD]

Hi all,

A word of warning to all NextSeq users about the new software update.

Our NextSeq 500 prompted a software update last week, which I did, and it worked just fine (I was running 1.2, and I updated to 1.3.0). However, what Illumina failed to communicate is that there are changes in the volume and concentration of the library sample that get loaded with software version 1.3. I only found this out by stumbling across the info below after overclustering a run and trying to figure out what had happened.

Illumina posted this information on their NextSeq FAQ page:


What is the library loading concentration required for sequencing on the NextSeq 500?

Using NextSeq Control Software v1.3, the library loading volume and library concentration are lower than with earlier versions of the software. The loading volume is 1.3 ml and the loading concentration is 1.8 pM.

With NextSeq Control Software v1.2, and earlier, the library loading volume is 3 ml and the loading concentration is 3 pM. However, the loading concentration can vary depending on library preparation and quantification methods.

Cheers,

Liz

[austinso]

Hmmm...that's a 4-fold drop in the amount of library that is being loaded...

How many reads are supposed to come out of that (low vs. high output)?

A.

[LizD]

I'm not sure how many reads, but we are getting about 50-60Gb from the Mid output kit and about 130Gb from the High output kits.

[JakobHedegaard]

Hi, updated to 1.3.0 yesterday but havnt conducted any runs on the NextSeq yet. Asked Illuminas Techsupport about the effected of the reduced loading volume and concentration on the yield. Their reply: "The same number of clusters will be generated leading to the same data output. This is achieved by differences in handling the fluidics which leads to a reduction in loss of library."
So leaving the library for a longer time on the flowcell to hybridize?
Anyway - as long as it works...
Cheers, Jakob

[LizD]

It definitely works!

[kentawan]

Hi folks! How's everything? Is it safe to convert to NCS 1.3.0 now? We are kinda scared after reading TS's comment and still stuck to 1.20 now.

By the way, what are the library concentration that you guys aim for when loading into the NextSeq 500? I realise that it is extremely difficult to be consistent in aiming for the right cluster density. In our lab we always aim for 1.75 pM but things always screw up and we often get 200+ k/mm² which led to poor sequencing results.

[ymc]

How's the v2 chemistry? Did it improve the quality significantly?

[Brian Bushnell]

How's the v2 chemistry? Did it improve the quality significantly?

I heard it was supposed to come out in March...

[kentawan]

I heard it was supposed to come out in March...

I called my local distributor. He mentioned that shipment starts February 2015!

[Brian Bushnell]

Oh, probably March is when we will first receive it, then. I'm very interested in whether, as claimed, it can reduce the error rate to on-par with HiSeq. Until that happens, it will remain a niche product here.

[ymc]

I found a public dataset called "NextSeq 500: TruSeq PCR Free WGS_RTA2.1.3.0 (NA12878)" at basespace. Does anyone know whether it is v1 or v2?