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[this question is based on a question that was asked on Reddit]

We're interested in doing whole-transcriptome cDNA sequencing of 24 mouse cell lines, and are deciding between Illumina and Nanopore sequencing. What are the costs involved in Nanopore sequencing, and how much hassle is it to analyse Nanopore data?

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The standard PCR-cDNA barcoding kit is \$750 USD, and has enough reagents for six runs, with each run using up to 24 samples (i.e. 144 samples total). Each run also needs one MinION flow cell (\$900 USD).

The total cost including external reagents is naturally dependent on the number of samples, and gets cheaper as the number of samples increases. For 24 samples, it's about \$5200 NZD (\$3100 USD). See below for more details.

If you could outsource this to a lab that has a PromethION (or P2 Solo), the sequencing would be a great lot cheaper. PromethION flow cells start at \$3600 for a pack of 4 (i.e. the same unit cost as MinION flow cells), but have about ten times the yield of MinION flow cells (which means more samples, or greater depth).

From a bioinformatics perspective, there's more work to do with nanopore reads because there are no commonly used / established pipelines (so I've had to create my own), but the results are much cleaner once they've been processed.

Cost Breakdown

For cDNA sequencing in Aotearoa, MinION works out cheaper than Illumina when compared against a service centre, about two thirds the price for us. Here is the reagent list I wrote up a couple of months ago for sequencing from 30 whole transcriptome samples, mouse or human (prices in NZD). This is with an ideal read threshold of 1M reads per sample (which I have found to give good quantitative results for differential expression). You could probably get away with lower yield for microbial sequencing:

ONT reagents:

  • 1 x PCR-cDNA Barcoding Kit 24 \$750 USD [\$1210 NZD]
  • 1 x Rapid Adapter Auxiliary V14 \$288 USD [\$481 NZD]
  • 1 x R10.4.1 flow cell @ \$900 USD [\$1503 NZD]

Total ONT cost: \$3194

Other reagents:

  • 10 mM dNTP NEB N0447 [\$145 NZD]
  • Maxima H Minus RT ThermoFisher EP0751 [\$346 NZD]
  • RNAclean or AmPure XP beads [AmPure XP: \$800 NZD]
  • Exonuclease 1 NEB M0293 [\$160 NZD]
  • RNAseOUT ThermoFisher 10777019 [\$569 NZD]

Total external reagent cost (excludes nuclease-free water, tubes, pipette tips, gloves, ethanol, DNA QC): \$2020 NZD

[As well as whatever you need to extract 200ng total RNA per sample]

Our current protocols for this PCR-cDNA barcoding + V14 RA approach can be found here.

The comparative Illumina cost according to the service centre we asked a couple of months ago was \$185 NZD per sample + \$5000 NZD per flow cell (or $9440 NZDfor 24 samples).

For almost all of these external reagents the amount ordered is an absolute minimum (from memory I think most are something like 50 reactions), so doing a second equivalent experiment would mostly only need the flow cells to be purchased.

In addition to the cost, turnaround time with MinION is quicker (for us it's a couple of weeks versus a couple of months for Illumina [with most of that time spent waiting in a queue]), and the results are nicer to analyse: full length transcript sequences with very even coverage profiles.

I'm aware that sequencing is more expensive in Aotearoa than it is in other countries, so the cost / time difference may not be as extreme elsewhere.

Bulk discounts for ONT flow cells

The prices above are for ordering reagents and flow cells one at a time, using the most expensive reagents. I call this a "grant funding" budget, because it's the quote I would give to people who are looking to fund research. There are ways to reduce costs when organising sequencing in bulk, and I've already hinted above at the extra capacity in reagent kits.

However, the biggest savings for nanopore sequencing can be achieved by ordering lots of flow cells at once. For example, ordering 48 MinION flow cells means that the per-flowcell price is \$500 USD instead of \$900 USD. That's 48 flow cells spread over the course of an entire year, with costs paid on delivery. If you can encourage multiple research groups to put in a combined bulk order, it's not too far out of the realm of possibility.

The long-read advantage

I've been told that a researcher is able to get 50M paired-end Illumina reads from a sequencing service centre for about $100 CAD.

50M paired end reads is about 10 Gb, which can be achieved on a MinION run (\$600 - \$1000), or about 1/10th of a PromethION run (\$850 - \$1500).

Even given those prices, it doesn't take much of a long-read advantage for MinION to become the most economical option. My own studies suggest that 1M full-length reads from nanopore sequencing (i.e. about 1/10th of a MinION R9.4.1 flow cell) gives results that are at least as good as 20M short paired-end Illumina reads.

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