Any reason to do MiSeq over NextSeq 2000?
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6 weeks ago
hjarnek ▴ 20

For less money than the MiSeq v3, the NextSeq 2000 generates more than 5x the sequencing depth, with the tradeoff being lower base quality. (I'm not sure how big the difference is though. As NextSeq is a two-colour technology, it calls G bases for no signal instead of N, so quality scores are more or less inflated and not directly comparable to MiSeq.) However, I presume things have gotten better in later years, and with the deeper sequencing you have more room to quality-filter your data. For my primary use case, metabarcoding, deeper sequencing also provides better opportunities for error correction, so in the end the difference in quality might be non-existant, I was thinking. Read length is not a deal breaker anymore either, as both platforms now offer 2*300 bp paired-end.

What are your experiences with NextSeq? Is the quality still a problem? If you have actively chosen MiSeq over NextSeq, what was your motivation? Is there really any case where MiSeq still has an advantage in practice, other than if you have very few samples and don't want to deal with the erroneous G bases or binned quality scores?

miseq nextseq illumina metabarcoding • 430 views
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6 weeks ago
GenoMax 148k

Quality is not really an issue these days for most any sequencing applications (unless you are working with degraded/ancient/odd samples). With Q score binning you don't really get true scores anyhow.

NextSeq 2K should be a fine alternative to use. Many large sequencing centers (e.g. JGI) use sequencers with 2 color chemistry to do metagenomic samples.

That said, for difficult to sequence samples, MiSeq has been the outstanding sequencing champ. No other sequencer performs like it. There is a new model of MiSeq i100 that was just announced that uses XLEP chemistry (2 color). We will have to see how it behaves compared to the vintage MiSeq.

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It would be interesting if someone had some NextSeq 2000 quality graphs to share (preferably 2*300 bp). I understand that a four-colour technology like MiSeq is hard to beat in terms of base quality, but base quality isn't everything. Especially in amplicon sequencing, better sequencing depth also means better error correction. And the patterned flow cell technology also offers greater sensitivity to rare variants compared to MiSeq (Singer et al, 2019).

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amplicon sequencing

Be sure to include adequate phiX when sequencing. Your sequencing center should know what to do but let them know that you have low nucleotide diversity samples (amplicon) when you submit.

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