Anyone use NextSeq or NovaSeq for 16S sequencing?

Hi all,

I know that NextSeq is only for short amplicons, ~150bp or around there, but I am still curious if anyone has used it for 16S sequencing? Nextseq gives around 120 million reads back compared to a Miseq for a similar price.

However, I know NovaSeq can handle larger amplicons and it gives back more than a billion reads. But I have not seen anything on people using it for 16S sequencing. Anyone know why? Seems way more cost-effective.


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Hello Sam,

Great questions!

I don’t run a sequencing core, but I’ve worked closely with folks who own both the Illumina MiSeq and NextSeq 550.

The Illumina MiSeq and NextSeq 550 are really different machines.

I think the MiSeq is commonly used for 16S analysis because of it’s longer reads (300 bp, paired end) and sweet spot for an amplicon study. With ~300 barcodes and 15 Million reads per run, you would hopefully get ~50k reads per sample, which is a reasonable number for an amplicon study.

While the NextSet 550 offers many more reads, the EMP primers still just offer ~300 barcodes. (Other barcoding schemes let you process more!) Plus, it might be preferable to sequence a longer region for better taxonomic resolution, than simply increase read depth. 300 bp > 6 million reads per sample :man_shrugging:

Oh, and buying one MiSeq is less expensive than buying one NextSeq 550 :money_with_wings:

But the NextSeq 550 is super reliable, and the MiSeq is ~11 years old.

So basically

  1. cheap machine, long reads, low depth, delicate
  2. expensive machine, shorter reads, greater depth, reliable

Are you planning to buy one of these machines, or learn more about the platforms before buying a run from a company or sequencing core?


P.S. If someone on here does run a sequencing core, please correct my mistakes! I would love to learn more about the NovaSeq, as I’ve not worked with one before!

Hi Colin,

Apologies on not answering this: my notifications were on a different email. Thank you for the detailed response. I agree with the differentiations between the NovaSeq and Miseq.

I thought it would be timely to leave a reply now however, as I am seeing that cores are offering NovaSeq 2x250 paired end sequencing at more reasonable prices. At 1.3-1.6B reads I think this could be huge for microbiome science. I am just not sure how well this has been validated for 16S sequencing.

If anyone on here has more info on that, it would be much appreciated!