no dual indices on NextSeq 500 (yet)

[SeqNerd]

There aren't a whole lot of NextSeq 500s in the wild yet, but for those lucky among you who have one, are expecting one, or hoping to run your libraries on one soon, please be aware that dual indices are not supported as of this writing. So pick your TruSeq/Nextera index combinations with care.

To quote Illumina over the phone: "we're working on it".

[BBthekid007]

Custom indexing is a no-go as well -- only 'official' Illumina index sequences can be used. As with dual indexing, they're "working on it".

One more note of caution: unlike the MiSeq, where longer-than-officially-supported reads are possible, the NextSeq will not allow runs that exceed the stated capacity of the reagent kits. We regularly do 350x300 or PE325 with the MiSeq's 600-cycle kit, but there's no way to do that with the NextSeq.

[nucacidhunter]

Quote from Illumina website:
"The TruSeq Dual Index Sequencing Primer Box supports the preparation of Nextera libraries for dual-indexed sequencing on the NextSeq 500, HiSeq 1000, HiSeq 2000, HiScanSQ, or Genome Analyzer instruments."

[avo]

We regularly do 350x300 or PE325 with the MiSeq's 600-cycle kit, but there's no way to do that with the NextSeq.

Do you do those 350x300 or PE325 reads with the standard 600-cycle kit, where the extra cycles derive from not reading indices and excessive reagents or is it some sort of "hacked" cartridge (like for the V2 kits)?

[bryanbriney]

Do you do those 350x300 or PE325 reads with the standard 600-cycle kit, where the extra cycles derive from not reading indices and excessive reagents or is it some sort of "hacked" cartridge (like for the V2 kits)?

We've done 350x310 runs with a standard 600-cycle kit, including an 8bp index read. Based on the amount of reagents left after a 350x310 run, you could probably go up to 350x325 with an index.

[avo]

Did any of these runs use the new MCS Version 2.4.1.3?
To me it triggers an error that the number of cycles is out of range for the used cartridge.

Maybe the number of cycles is now limited in the new version of MCS?

[supuamina]

I would love to hear more feedback on anyone who currently uses a Nextseq 500. What are the likes and dislikes of this instrument?

[bryanbriney]

We've done about two dozen runs and have been really happy with our NextSeq. Total throughput regularly exceeds Illumina's specs (not uncommon for us to get ~500M reads in high-output mode). We also have MiSeqs, and read quality between the two instruments is roughly similar -- we only do PE300 reads on the MiSeq, so we can't do a direct quality comparison. In our case, which involves sequencing low diversity libraries, we haven't had any issues with the 2-color vs. 4-color sequencing.

Any specific questions you'd like me to address?

[supuamina]

What are the Q scores of the sequences coming off the NextSeq 500 compared to those of MiSeq. What are the error rates of the NextSeq 500 so if looking for a MAF>0.5% in an Onco sample?

[Brian Bushnell]

You may want to look at this thread. The NextSeq quality scores are not currently comparable to MiSeq/HiSeq.