Low diversity on Nextseq 500

[MisUser]

Hi all,

Does anyone have already sequenced low diversity libraries on the Nextseq and how did it perform? What were the conditions (PhiX % and library quantities)?
How many reads did you get?
Thank you for your input!

[jteeee2]

What kind of libraries are they? Are you expecting diversity to be low throughout the entire read length or just in specific parts?

We have had success with certain types of low diversity libraries on the NextSeq. Specific performance will depend on the exact library type, but a good place to start would be a PhiX spike-in of 10-20%.

-JT

[MisUser]

Thanks for your quick reply.
The sequence is identical for the first 25 nts and the diversity is good for the next 20nts.

[jteeee2]

We haven't dealt with this specific situation before, but with that length of identical sequence I would probably aim a little higher with the PhiX for the first attempt (20-30%). Would be interested to hear if other folks have sequenced this kind of library on the NextSeq. Once you convince yourself you can get good data from the high PhiX spike-in, you can titrate it down on future runs so you don't waste so many reads.

[cmbetts]

I think that 25% is a pretty good starting number. I used to regularly sequence libraries that looked like (8bp diversity + 15bp linker) x 3 on a NextSeq, and things tended to go badly when we dropped the PhiX below 20%

[nucacidhunter]

NextSeq seems to perform good with low diversity and unbalanced libraries such as RRBS as shown in the following:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5065634/

[dlin82]

Hi - sorry to revive an old thread, we are gearing up our own very first RRBS run on the NextSeq500 so wanted to check in if 30% is actually a good amount of PhiX to spike in. Are the quality of clustering consistent?

Consulting with Illumina and other sources they suggest a starting amount of 40-50% (more like 50%) PhiX, and you really do lose quite a lot of reads with that amount so I would like to avoid that at all if possible. Thanks so much everyone!

[nucacidhunter]

Two issues with RRBS sequencing:
1- unbalanced base composition: NextSeq performs fine
2- low diversity at the start of reads: depends on method and if you are using NuGEN kit 1% PhiX would fine but if you are doing end repair, A tailing and ligation based method then 20% PhiX spike in is good starting point

[dlin82]

Thanks for the quick reply nucacidhunter!

Yes it's homebrew so I guess I'll start with 20-30% PhiX spike-in. I am reading the Meissner mRRBS paper more in detail and noticed they do dark cycles. Does it help in this case (or can the Nextseq even do this)?

Thanks so much again, I am basically thrown into this NGS business with zero previous training and am still trying to figure things out!

[nucacidhunter]

For dark cycles you would need Illumina's assistance in your own risk if they agree and generally is worthwhile if you are doing multiple runs. I have not heard of dark cycles on NextSeq.