Next Generation Technologist

Next Generation Sequencing, Marketing, and the Genomic Revolution

March 13, 2014
by Dale Yuzuki
1 Comment

Ion Chef™ System ships to first customers

Ion Chef Shipments going out (photo credit to Michael Aken of Life Technologies)

Ion Chef Shipments going out (photo credit to Michael Aken of Life Technologies)

Way back when I was pouring 35S-labeled dNTP Sanger sequencing polyacrylamide gels (and fond memories of using reagents like degassed acrylamide, TEMED and Silane), robotic automation was at that time only in an industrial or manufacturing context. Now there are many automated liquid handling companies (for example Beckman is a popular choice, but Tecan and Hamilton and others share the market), and many options when it comes to setting up reactions in a 96-well format.

But when it comes to taking a single next-generation sequencing library molecule, affixing that molecule to a bead or particle (or in the case of a 5500xl Wildfire or Illumina flowcell) and then amplifying that molecule 1000′s or 100′s of thousands of times is not a trivial task. This is typically termed ‘template preparation’ – alternatively Illumina calls it ‘cluster generation’. Continue Reading →

March 11, 2014
by Dale Yuzuki

Some clarifications about Ion Torrent PII and NextSeq 500

Yesterday’s Ion Torrent Proton PII™ and Illumina NextSeq 500™ post certainly got a reaction from several quarters, including detailed pricing information about the 1x75bp format for the high-throughput configuration on the consumables.

Instead of making edits to the original here are some clarifying points, as it is clear that Illumina is making a break from their prior pricing model that they used for the MiSeq.

For those not familiar with MiSeq pricing, to compete with the Ion Torrent PGM™ relatively inexpensive 314 and 316 chips (about $300 and $550 per run respectively at a current >60MB and >600MB throughput respectively), Illumina has a ‘micro’ and ‘nano’ sized flowcell / reagent kits with lower throughput, that have similar read numbers and throughput. However, due to the cost of the reagents and flowcell, the smaller-sized kits are still expensive to produce and to sell, with the least expensive ‘micro’ kit still costing on the order of $800 or $900 to run.

Thus I mistakenly assumed that a 1×75 high output run on the NextSeq 500 would be priced relatively high – I used $3500 per 1x75bp run for the ‘High Output’ mode – which was incorrect. Based upon some pricing information I was given, the reagents were not just 20% less but a full 66% less – one third the price I had originally surmised, or $1300 per 1x75bp run.

Also on the point of 400M reads in the high output mode, it is unfair to use the 400M read number when it is clear in Illumina’s documentation that the number is ‘up to 400M’ reads (referring to their spec sheet).  Reading closer, the throughput on 1×75 is 25-30GB in high output mode, and 25GB / 75bp = 330M reads. So using 330M reads at $1300 / run gives a $393 per 100M reads metric.

Doing a similar calculation for the Mid Output, 2x75bp configuration (and was told that that pricing is around $1000 per run) at 16GB, the calculation comes out to 106M reads at $1000 / run equating to $943 per 100M reads.

So the pricing per 100M reads on the high output mode in a 1x75bp configuration is aggressive pricing, sitting right on top of where the PII is. Doing other calculations for throughput (price per GB) appears to be much more in line with the prior model (i.e. fewer cycles = less efficient on from a cost per GB perspective).

And so I present to you and updated chart, having to add a large number of rows to accommodate all the different pricing permutations. For those of you who have never worked in marketing within a commercial life science research market company, the two most difficult things you will ever work on are naming products, and pricing them. I know the meaning of the phrase first-hand: “No one knows the price of anything”, because of course the price of anything is what individuals are willing to pay.

Revised version of a PII and NextSeq 500 comparison chart

Revised version of a PII and NextSeq 500 comparison chart






















So the picture is not nearly so clear-cut in terms of pricing – and looking over the ‘cost per $100M reads’ for the different formats on the right, that $393 pricing does appear to be somewhat incongruent.

It is clear from this however that the Ion Proton with the PII will be less expensive to run, regardless of the mode (on the order of 30%, the difference between a $1,000 PII run and a $1300 1×75 High Output one), and less time to get data (8h vs. 11h), and on-par in terms of cost per 100M read metric.

I learned something yesterday – and every day that I learn something is a good thing. The NextSeq chemistry uses a fluor for ‘T’ base, a different fluor for the ‘C’ base, and the ‘A’ base is composed of two fluors that have similar emission / excitation spectra as the two prior bases but are not identical. Why this is done very likely resides in some complex explanation, perhaps involving biochemistry (the ability of a modified polymerase to accept a nucleotide decorated with two different fluors must be a wonder to behold at the atomic level), or optics (there is an incredible amount of engineering involved when you are using a system that started out with the first Solexa instruments basically a confocal microscope scanning device). Thinking through the chemistry, and how different it is compared to the existing HiSeq / MiSeq chemistry, it will be of interest to see what kind of GC sequence bias it has along with the primary modes of error.

On that note I’ll have to start searching for a NextSeq dataset and play around with it.

Way back when I first joined Life Technologies in early 2010, a person involved at Sanger during the Human Genome Project (HGP) days said during a SOLiD 4 training “when there’s money to be made, miracles happen” regarding technology development intersecting a free market. That point rings true again and again. It was last year during a talk Eric Green (Director of the National Human Genome Research Institute) was giving where he wondered whether the cost-per-MB curve that the NHGRI NISC regularly publishes had hit a plateau or not. It’s clear that market competition will be forcing prices down yet again.

March 10, 2014
by Dale Yuzuki

The upcoming Proton PII and the NextSeq 500

Record PI runs with a 20.5GB at the top, from the Ion Community site

Record PI runs with a 20.5GB at the top, from the Ion Community site

There has been a lot of publicity around the NextSeq 500 from Illumina, and it appears to have been designed to compete directly against Ion Torrent’s upcoming PII chip. Thanks to a visit to upstate New York last week, I met Dr. Sridar Chittur who told me how important it was to put current information out on this blog, and if I can put out the disclaimers up-front it would be very helpful for those thinking about what benchtop system to purchase over the next several months. Continue Reading →

March 3, 2014
by Dale Yuzuki

Nabsys single molecule mapping technology

Close-up of Nabsys ChipAnother interesting single-molecule technology is a company out of Providence (RI) called Nabsys. For several years I had heard the name involved in developing single-molecule sequencing technology, and this technology will start its initial product around genomic mapping, rather than sequencing.

For background on genomic mapping and CNV analysis along with the competitive landscape, here are  prior pieces written previously called BioNano Genomics, Opgen and Copy Number Variation, and an update on BioNano from last Fall’s ASHG meeting. So while BioNano Genomics and OpGen both use optical mapping of single molecules, Nabsys uses electrical detection. (Cue the optical vs. digital detection methodology of Ion Torrent here). Continue Reading →

February 24, 2014
by Dale Yuzuki
1 Comment

Targeted RNA Sequencing Approaches

Happiness is getting more of what you want, in RNA-Seq as in other things...

Happiness is getting more of what you want, in RNA-Seq as in other things…

There are several commercial methods for looking at 10′s or 100′s of gene expression levels via a high throughput TaqMan™ assay from Life Technologies / Thermo Fisher Scientific, a competitive offering from Roche, Douglas Scientific, or also Fluidigm. The limitation of these technologies however is the amount of multiplexing a single assay in a given volume, which regardless of the amount of miniaturization does limit the samples by genes evaluated throughput.

To perform RNA-Seq, one looks at all the particular RNA species present, dependent upon the up-front sample preparation. (To clarify, a miRNA experiment would purify small RNAs then go into cDNA synthesis and sequencing; mature polyA+ RNA can be purified and then cDNA made and sequenced etc.) But what about a targeted set of expressed genes to evaluate via NGS? Continue Reading →

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