[TonyBrooks]

This upgrade really is a pain in the backside. I have no idea why Illumina couldn't make this back compatible and then use the RFID chips to determine whether to process the run as required.

I wonder what happens if someone comes wanting to compare to an older data set once we've switched to v2. They'll need to pay to re-run those old libraries on v2.

[SA2002]

Hello,

Has anyone used v2 reagents with NCS v1.3? If so, what did the results look like?

[TonyBrooks]

Hello,

Has anyone used v2 reagents with NCS v1.3? If so, what did the results look like?

I'm pretty sure you can't do this as the software would surely check which kit version you are using and tell you it's incompatible.
It also looks like they changed the dyes, so the lasers would most likely excite at the wrong wavelength.

[GenoMax]

Hello,

Has anyone used v2 reagents with NCS v1.3? If so, what did the results look like?

Quote from here:http://support.illumina.com/sequenci...xtseq-500.html

NCS v1.4—Required for use with the NextSeq 500 Kit v2. NCS v1.4 is optimized for v2 reagents. Earlier kit versions are not compatible.

NCS v1.3—Compatible NextSeq v1 reagents.

[SA2002]

Thanks for the replies. Actually we recently ran kits with v1.3 but noticed that the stickers on the flow cells looked slightly different from previous flow cells and we got low density clusters. We're in the process of trying to figure out whether our reagents were v1 or v2 and was just curious if anyone else ran into the same issue.

[rogerzzw]

WGBS poor index and read2 on Nextseq

Hi, Guys

I am new to NGS. We've been trying to do whole genome bisulfite sequencing with Nugen Kit on Nextseq 500 for a few times with 2 samples.

We always encountered the same problem. After demultiplexing, there is about 50% of unknown index reads (most of them are "GGGGG" and "NNNNNN") . According to other centers using exact same kit and machine, they do not have any problems with adapters and indexes. And we always use single-use illumina kit and reagent for each run. So we do not know what happens.

We ve also tried to map read1 and read2 (with unknown barcodes). The alignment of read1 is quite good (90% aligniable), but reads2 is not good (~40%).

We used custom primer for read1 and illumina primers for read2 and index.

Do you guys have some ideas on what happened?

Many thanks in advance!

Roger

[GenoMax]

@rogerzzw: I assume you have been in touch with Illumina tech support about this? What was their take?

In general tag read issues with other illumina sequencers (don't have direct NextSeq experience) happen when a sample is (borderline) overloaded. Has that possibility been eliminated?

[rogerzzw]

@rogerzzw: I assume you have been in touch with Illumina tech support about this? What was their take?

In general tag read issues with other illumina sequencers (don't have direct NextSeq experience) happen when a sample is (borderline) overloaded. Has that possibility been eliminated?

Hi, GenoMax

I talked to tech support. We loaded as suggested (1.8pM - 2pM), but our cluster density is always lower than expect( ~140k/mm2 vs ~200k/mm2).

I learnt from several posts that the tag reading may be due to overloaded. But in our case, we actually turned out lower density, which makes me very confused.

What do you think?

BTW, illumina said it is due to low diversity libraries. But I knew there are few groups only spike in 5% Phix and got quite good results.

Appreciate!

[GenoMax]

Were your runs using V1 or V2 reagents? There is significant differences between chemistry and run procedures between those versions. Hopefully someone with direct NextSeq experience will be able to chime in soon.

[rogerzzw]

Were your runs using V1 or V2 reagents? There is significant differences between chemistry and run procedures between those versions. Hopefully someone with direct NextSeq experience will be able to chime in soon.

We always use V2.

Our machine is brand new, and it had a good 1st run only on Phix. Then I started to see the tag reading problem for 4 times.

Once comparing with other people's method of library prep, we found no difference between two. But we always is quite far from expectation.

[kentawan]

Were your runs using V1 or V2 reagents? There is significant differences between chemistry and run procedures between those versions. Hopefully someone with direct NextSeq experience will be able to chime in soon.

V2 is superb! One of our run uses the same library, once on the V1 and another on V2. The V1 run's cluster density was about 170(sorry i forgotten the exact unit, while V2 was 198.

Our V1 run gave us q30 passing of about 78% while the V2 gave us 92%! Despite the higher density cluster!

[rogerzzw]

V2 is superb! One of our run uses the same library, once on the V1 and another on V2. The V1 run's cluster density was about 170(sorry i forgotten the exact unit, while V2 was 198.

Our V1 run gave us q30 passing of about 78% while the V2 gave us 92%! Despite the higher density cluster!

Hi, Kentawan.

I am a beginner of NGS on Nextseq 500. I am primarily doing whole genome bisulfite sequencing with Nextseq 500. I saw your post on this forum and would like to learn from you.

1. based on what you said, you use V2 kit and got a very good cluster density. we used V2 but never get that high cluster density. Ours is about 140K. Can you share your experience on that? Appreciate!

2. We always had a big issue on Barcode reading, other metrics look very good. There are a high portion (35%) reads are reading as "GGGGGG", I think it is due to reading failure, but we do not know how to pin it down, we pooled two samples each time. Unfortunately, there was one time, we pooled with other 4 Chip-seq samples and still the same. Do you some idea how to fix it?

Thank you very much

Roger

[GenoMax]

There are a high portion (35%) reads are reading as "GGGGGG", I think it is due to reading failure, but we do not know how to pin it down, we pooled two samples each time. Unfortunately, there was one time, we pooled with other 4 Chip-seq samples and still the same.

Roger

That is not good news. That would mean there is something wrong with your bisulfite libraries since low nucleotide diversity should not have been an issue with 4 additional samples (these additional 4 samples had diverse barcodes?).

[rogerzzw]

That is not good news. That would mean there is something wrong with your bisulfite libraries since low nucleotide diversity should not have been an issue with 4 additional samples (these additional 4 samples had diverse barcodes?).

Yes, those 4 Chip-seq samples barcodes are well selected. But, the problem is I am not sure if it is due to low diversity since it is highly diverse when we combine chip-seq samples. It was still problematic when demultiplex, pretty much similar to the trial when we only have 2 samples for bisulfite sequencing.

[rogerzzw]

@GenoMax

if it is library issue, 60-70% are demultiplexed very well, I cannot explain that.