Illumina get small with new miniSeq and FireFlight

After bullying the NGS market a couple of years ago with its new large sequencing platforms able to deliver thousands of human genomes per year, now Illumina decided to further expand its offer with also small solutions.

Few days ago at JP Morgan the company revealed a new benchtop sequencer aiming at small labs and clinical application based on gene panels. The new machine, called miniSeq, can produce up to 8 Gb per run with 25M reads, costs "only" ~50k$ and promise a cost per run between $200 and $300.
With this move Illumina try to challenge Ion Torrent PGM and the Thermo Fisher good position in the area of small, rapid an cheap sequencing. The new sequencer is based on the two-color technology developed for the NextSeq and HiSeq X sequencers allowing to reduce machine cost and speed up the sequencing runs.

Look how it compares to the existing Illumina benchtop sequencers:

In its try to fill all the space in the market Illumina has also annunced an even smaller sequencer, developed under the name of "project FireFlight". The details released included a 1.2Gb output, one "colour" SBS and patterned flowcells. It also has a digital fluidics library prep module. The machine alone might cost just $15,000, with under $200 cost per run. The new machine will be based on semiconductor technology with CMOS sensor collecting light data from multiple simultaneous reactions. Project Firefly will be developed through 2016 and expect to deliver in 2017.

More details and interesting consideration have been provided on the Illumina website and other omics blogs, like CoreGenomics and OmicsOmics.

Main new technologies from AGBT15

Ion Torrent

• The new avalanche chemistry.

This new chemistry based on isothermal amplification will provide longer reads up to 600bp on PGM an 400bp on Proton PI with lower costs for template preparation. 

• New protocols for Ampliseq library preparation on Ion Chef system. 

The company aims to transfer all the processing steps on the new Ion Chef that will beacm the all-in-one solution from library prep to chip loading, minimizing hand on time. Take a look to the video on you tube.

• News on the PII chip.

They finally reported about a full working version of the PII chip producing up to 300M reads with 100bp length. However an official release date was not announced

Illumina

• NeoPrep, the new automated system for library preparation. 

Having already presented some new sequencers few months ago, Illumina come to the stage with this new equipment that will provide fast and accurate library preparation and can prepare 16 libraries at with as little as 30 minutes of hands-on time. NeoPrep is based on the electrowetting microfluidic technology and use A cartridge with reagents and your sheared DNA. The instrument generate and quantify each library, ready for pooling. Roughly is required per run. All this stuff at the price of $49K ($39K introductory pricing for the first 6 months). More details at the Illumina page or on Omics Omics blog.

10X Genomics

10X Genomics is the real innovation this year. They revealed their new GemCode technology to reconstruct long reads and haplotypes from standard short reads sequencing. The new instrument will integrate in the standard Illumina based library preparation, thus configuring as an add-on for already equipped laboratories. Using dedicated pen source software and genome browsers from 10X Genomics one can then generate standard file formats (such as phased VCF or BAM with phase tags) and visualize the reconstructed haplotypes. 

Take a look at this nice video or read more details from Omics Omics. 

First user reports on Oxford Nanopore MinION!

After the start of the early access program, the sequencing community is waiting for the first results and comments on the MinION platform by Oxford Nanopore. This sequencer promises to revolutionize the field and is the first nanopore based sequencer that have reached the market.

Nick Loman, one of the early customer has now reported the first results obtained on the new platform. It is a 8.5 Kb read from P. aeruginosa showing that MinION can produce useful data, even if the accuracy remains low. Analyses of a read by two bioinformatics researchers, who used different alignment tools and posted their results here and here, showed that the read is about 68 percent identical to the P. aeruginosa genome and has many errors, particularly gaps. Main issues seems to be in the basecalling software, but Oxford Nanopore is working hard to improve it. Accordingly also to Konrad Paszkiewicz, another early customer, the device itself is really robust and easy to use and the library preparation procedure is simple, resulting in low sequencing costs.

The mean read length seems to be about 10 Kb, but users reported even longer reads up to 40 Kb, covering the entire lambda genome used for testing. So the read length is really promising and place the mature MinION as a natural competitor to PacBio.

The use of MinION seems straightforward: after plugging the sequencer into a USB 3.0 port of a computer, it installs the MinKnow software suite. A program called Metrichor uploads the raw data – ion current traces – to the Amazon Elastic Compute Cloud, where base-calling happens, either 1D base-calling for unidirectional reads or 2D base-calling for bidirectional reads.
Overall, improvements have to be made to the base-calling software, reliability of the flow cells, and library shelf-life, and new software needs to be developed by the community to take advantage of the MinION reads.  Oxford Nanopore said a new chemistry will be available in the coming months, which might include some of these improvements.

In the meantime, many other early access users contacted by IS website are awaiting the arrival of reagents, are in the midst of burn-in, or have run their own samples but are not ready to talk about their results yet. So we are expecting many more data and comments and detailed estimation on platform accuracy and data production to be out in the next months! The new minion has fulfilled the excpection in this first test and there is a lot promising about this new technology...maybe a new revolution in the field is ready to come!

Other details can be found on this post on GenomeWeb.

Genapsys reveals GENIUS at AGBT

At the last AGBT meeting Genapsys, a small company from California, presented its new "launch-box" sequencer called GENIUS (Gene Electronic Nano-Integrated Ultra-Sensitive).

This machine, with the size of a toaster, is a new kind of NGS sequencer that apply electronic sensor technology and promise to produce up to 100 Gb of sequences in few hours, with read length up to 1000 bp. Like the MinION they propose to push the NGS market even further, providing a new generation of sequencher which are incredibly small, cheap and easy to operate. A step further to the so called "freedom of sequencing".

Genapsys started an early-access program for the GENIUS platform last week and plans to ship out the first instruments within a few months, followed by a general commercial launch either this year or next year.

The system has an opening in the front to insert a small, square semiconductor-based sequencing chip. A reusable reagent cartridge attaches to the back, and a computer for data processing is integrated. Based on the Genapsys CEO interview at InSequence, library preparation require clonal amplification of the template DNA on beads in an emulsion-free off-instrument process that requires no specialized equipment from the company. While the first version of the Genius will require separate sample and library preparation, the company plans to integrate sample prep into the platform eventually. The template beads are then pipetted into the chip, which has "millions of sensors" and a flat surface with no wells. There, the beads assemble into an array-like pattern, with each bead being individually addressable. This is followed by polymerase-based DNA synthesis where nucleotides are added sequentially. The system uses an electronic detection method to identify which nucleotide was incorporated, but the detection principle remain secreted by now, even if it is not based on pH measurements (like the Ion Torrent technology).

Three types of chips will be available for the Genius, generating up to 1 gigabase, 20 gigabases, or 100 gigabases of data with the sequencing run taking only few hours. Estimated sequencing costs per gigabase will be $300 for the smallest chip, $10 for the middle chip, and $1 for the largest chip. Even if the instrument price has not been determined yet but, Genapsys will provide special offers to customers committing to high usage. This will be something like an usage plan that include the instrument at a lower price, but with a minimum expense in consumable per year. Esfandyarpour, Genapsys CEO, said they have already generated sequence data on the platform, though he did not provide specifics. The data quality will be "equivalent or better than the best product out there," he said.

Illumina presents two new sequencing platforms: population scale genomics and the 1000$ genome

Few days ago Illumina announced two new NGS platforms: a huge factory scale sequencer called HiSeq X Ten and a new benchtop sequencher called NextSeq 500, half-way between a MiSeq and a HiSeq.

Both platforms represent a huge advancement in data production, made possible by several technical innovations and a new chemistry. First of all, Illumina worked hard to increase accuracy and speed of image acquisition using an increased number (up to 6) of new LED cameras for image snapshot and a new flow cell design with larger random clusters enabling it to work with the lower-resolution optics as well as new surface chemistry to enhance the signal.

The HiSeq X Ten will also integrate a dual direction image scan system dubbling the scan speed and a new flow cell containing nanowell that allow for a precise cluster separation resulting in more dense clustering.

Both instruments will run with the new 2-color chemistry. This methods use only 2 different fluorescent molecule, red and green: T and C bases are marked as green or red signal, respectively; A is marked with both signal and G lack any marker. Thus, only 2 image acquisitions, one per color channel, are needed every cycle, instead of the classic 4, cutting down the processing time. The chemistry is well explained in CoreGenomics blog and the Illumina tech sheet.

  

The NextSeq 500 will come together with two diffent flowcell and two different run mode, resembling the fast run mode of the HiSeq 2500.

The mid-output flow cell includes 130 million clusters and will support a 2x75 base kit that will generate 16-19 gigabases of data per 15-hour run, or a 2x150 base kit that will generate 32-39 gigabases of data in a 26-hour run.
The high-output flow cell includes 400 million clusters and will support a 2x150 base kit that will generate 100-120 gigabases of data per 29-hour run, a 2x75 base kit that will generate 50-60 gigabases per 18-hour run, and a 1x75 base kit that will generate 25-30 gigabases per 11-hour run.

This numbers will allow a WGS sequencing in little more than a day at about 30X, making the NextSeq 500 the first benchtop sequencer to hit the goal of whole genome.

The HiSeq X Ten is a huge sequencher and it's actually composed by 10 single sequencing unit that will cost you a total of 10M$. Illumina will accept only a minimum order of ten units, with each supplementary unit costing 1M$. One unit will be able to generate 600 gigabases of data in one day, enough to sequence five human genomes, or 1.8 terabases of data in under three days, so that the total data production will be 18Tb every 3 days, allowing the sequencing of 18000 genome every year!!

Illumina claims that this juggernaut will respond to the need of population scale sequencing programs, often national health programs, such as UK initiative to sequence 100000 individuals or the Denmark project to sequence the entire population of an isolated island.

The HiSeq X Ten will enable the "first real $1,000 genome," said Flatley, CEO of Illumina. One reagent kit to support 16 genomes per run will cost $12,700, or $800 per genome for reagents. Hardware will add an additional $137 per genome, while sample prep will range between $55 and $65 per genome.

However, the new machine will sequence ONLY whole human genomes, no other applications are supported by now, and, given the hard work needed to produce and set up such a huge instruments, Illumina will deliver only 5 of them in the first year.

Despite the 10M$ price, Illumina has already sold 4 HiSeq X Ten: to the South Korean sequencing service provider Macrogen, the Garvan Institute in Australia, the New York Genome Center, and to the Broad Institute, which purchased a 14-unit system.

Detailed information and interesting discussions around the two new platforms and their technical innovations can be found around the web: CoreGenomics (presentation, HiSeq X Ten, NextSeq 500), MassGenomics, Omics!Omics!, Opniomics, GenomeWeb, Nature News. 

So is the mythical goal of 1000$ genome finally achieved? Well, it seems almost...

First of all, one have to consider the initial investment and the overhead costs to run the 10 machines. Moreover, the cost estimate made by Illumina are based on 4 year full activity of the HiSeq X Ten, which means 18.000 genomes per year per 4 year with machine running 24h/day...This scenario seems unlikely to many experts, since we simply don't have so many samples to sequence.

Finally data analysis costs, besides the simple sequence alignment and maybe SNP call, are not included as usually. For a more detailed evaluation of the real costs, read the interesting post on allseq blog.

Ready for Avalanche?

It's been a while (I remember rumors more than 1 year ago) since Life Tech started talking about a new chemistry based on isothermal amplification that promise to eliminate emPCR, shorten clonal amplification down to less than 1h and provide better uniformity...They called it Avalanche...But no more has been revealed in the past months, such that Avalanche was becoming like a mythological creature: sure it's fascinating, but does it really exist?

However keep your new stuff secret since the time of commercial release seems to be a common way of acting in the competitive field of NGS technology...and now finally Avalanche is ready to hit our bench and provide the long waited improvement for SOLiD and Torrent sequencing platforms.
Indeed, researchers from Life Technologies has recently published on PNAS a paper demonstrating the feasibility, efficiency and rapidity of the new method. As anticipated, it's based on isothermal amplification of a properly prepared DNA template using the bst DNA polymerase and substrate immobilized primers. Citing the abstract, it use "a template walking mechanism using a pair of low-melting temperature (Tm) solid- surface homopolymer primers and a low-Tm solution phase primer". Authors report results obtained with the new method applied on a SOLiD 5500 W flowchip and they are quite exciting: reaction time of slightly more than 30 min with high percentage of monoclonal colonies and 3- to 4-fold more mapped reads than with traditional method and easy paired-end protocol.

Since the procedure reported in the paper is developed on SOLiD technology, I expect that the new chemistry will be commercially available in short time on this platform...However, I'm wondering if Life Technology is already working to extend this innovation also to Torrent sequencers and if they will introduce Avalanche with the PII chip (announced to be out in these months) or with the PIII ( probably in the first half of 2014). The method demonstrated in the paper require surface immobilized primers on a flowcell, so they have to adapt it to work on the small beads that are required for the PGM and Proton chips...or they have to re-think the chips themselves to avoid use of beads, but I think this solution is not so easy to apply on the current sequencer machine. Another question that bother most of the Torrent cutomers: will Avalanche be compatible with the current library/template preparation equipment or we will have to buy some new expensive piece to actually do the upgrade?

Now waiting to be trampled by the Avalanche!

For more technical details read the full paper:
Isothermal amplification method for next-generation sequencing. 

Ma Z, Lee RW, Li B, Kenney P, Wang Y, Erikson J, Goyal S, Lao K. 

Incredible But True: Life Technologies introduces an amplicon-based exome sequencing kit

Life Technologies has launched an exome capture kit that makes use of its AmpliSeq technology.
According to the manufacturer, the AmpliSeq™ Exome Kit minimizes the high cost and complexity of exome sequencing enabling the enrichment and sequencing of ~294,000 amplicons (!!!).
The kit targets 97% of coding regions, as described by Consensus Coding Sequences

(CCDS) annotation, in 12 primer pools for highly specific enrichment of exons within the human genome totaling ~58 Mb (it is not clear to me if the amplicons include sequences like UTRs and miRNA).  The novel technology, designed for the Ion Proton platform, delivers >94% of targeted bases covered at 10x even with two exomes per Ion PI  chip. Total workflow from DNA to annotated variants of an exome can be achieved in two days, including six hours for exome library preparation and three hours of sequencing time. Compared to hybridization approaches for exome sequencing, one advantage of an amplicon-based approached is that the input amount is small (as little as 50 nanograms).
Additional information can be found in this Life Technologies' Application Note.

2012 will be the year of Nanopore Single Molecule DNA Sequencing

In a couple of weeks we will know more about it. It has been confirmed that Clive Brown, the Chief Technology Officer of Oxford Nanopore Technologies, will be talking at the AGTB meeting about “Single Molecule ‘Strand’ Sequencing Using Protein Nanopores and Scalable Electronic Devices”. On February 17th we will likely know if the new technology will represent a competitor of the (relatively) few but long reads of the PacBio RS or if it will be directely projected in the $1000 genome race.

You can read some interesting speculations on what is going on in the following posts on the Core Genomics blog: Oxford Nanopore confirmed at AGBT; Nanopore sequencing: is the hype about to end? How does a nanopore sequencer work?

Today InSequence has an article with an intriguing title on the same topic "Oxford Nanopore to Commercialize DNA Strand Sequencing this Year; Illumina not Involved".

A press release on the ONT website clearly states "Oxford Nanopore intends to commercialise DNA strand sequencing products, directly to customers within 2012. At the AGBT presentation, Oxford Nanopore will show DNA strand sequencing data and other disruptive features of the Company's proprietary electronics-based sensing devices."