Biotech Experts Highlight Clinical Sequencing at Rx/Dx Summits

I recently attended the IBC Rx/Dx Summits held in San Francisco in the first week of August 2012.  The meeting was held at the Westin San Francisco Market Street Hotel.  I was attracted to this event because it gave me the opportunity to learn about some of the new emerging market dynamics in next generation sequencing (NGS) and other areas that I track for my firm.

I listened to a talk comparing desktop sequencing systems by Jason Lih, Ph.D., Principal Scientist, SAIC-Frederick.  His talk was called Assay Development for Detecting Somatic Mutations in Cancer by Targeted Amplicon Sequencing: A Technical Comparison between PGM  and MiSeq.

Dr. Lih’s talk compared two desktop NGS machines, the Life Technologies, Ion Torrent, PGM with the Illumina MiSeq. At the beginning of his discussion, he said that he would not say which NGS platform is better.

In his NGS application, he used targeted amplicon sequencing to develop assays to detect somatic mutations in cancer.  Jason said that the PGM used AmpliSeq  v. Illumina’s TruSeq Custom Amplicon  (TSCA) technology.  He said that Life’s PGM requires just 20 ng of DNA sample, whereas the Illumina MiSeq requires 250 ng of DNA sample.  The Life PGM uses a 4-plex #‘316’ chip which outputs 1×200 base pairs of bi-directional sequence in one day plus 4 hours. (or 28 hrs).  The MiSeq takes 27 hrs (or 1 hr. less).

Using a comparison concept that he called the “Cosmic” MOI (Molecule Of Interest), he created a comparison chart comparing 1160 Cosmic MOIs.  He compared both vendor’s reagents.  His results showed that the PGM produced slightly more MOIs.

Vendor Model Reagents MOIs DNA Sample Run Time QScore
PGM AmpliSeq 1148 20ng 28 hrs 30
MiSeq TSCA 1108 250ng 27 hrs 30

The PGM variant caller was the Ampliseq Reporter.  He used a 3rd party software from CLC Bio.  The CLC Bio Integrated Genome Viewer showed a Qscore of 30 for each NGS machine.

What is interesting to me is that at end of his talk during the Q&A, an attendee asked Jason for his opinion about which was the best of the two NGS machines that he compared.  He said that his comparison was not intended to find the “best” NGS machine. My take away from his answer was that as far as Jason’s application was concerned, one could use either NGS machine and get comparable/ usable research data.  Also of note is that Roche Applied Systems demonstrated their 454 GS Junior desktop sequencer at the exhibit hall.  I wonder how the 454 GS Junior would compare against the PGM and MiSeq machines.

During the lunch- networking break in the exhibit hall, I met Robert Klein, Ph.D., Chief Business Development Officer, Complete Genomics, Inc. who said that he was giving a talk later in the day.  I attended his talk called: Large-scale, Accurate Whole Genome Sequencing to Enable Genomic Medicine. 

Robert gave a update on the business direction or activities at Complete Genomics (CG).  He said that CG v.1 was about research sequencing and that CG v.2 is more about clinical sequencing.  Dr. Klein said that in 2006 CG developed its proprietary sequencing technology and service model.  By 2011 they had delivered 3,000 genomes to customers.  Robert said that CG now produces 1,000 genomes per month.  He explained that they have a DNA factory in Mountain View and sends the data to its data center in the nearby city of Santa Clara.  CG does this because Santa Clara offers a lower cost for electricity.  CG provides “research ready” data to the customer and the customer analyzes the data.

Robert highlighted CG’s goals as including: Setup a CLIA facility 2H’12, Scale-up quality, Scale down cost, Scale-up throughput and Offering ‘clinical use’ sequencing.  CG will be focusing on new apps. including Idiopathic kids, Refractory cancers, Replacing cytogenetic arrays and Replacing targeted panels.  Dr. Klein also added that CG is interested in Wellness/ concierge medicine and Reproductive genetics.  He mentioned that CG is exploring other market spaces such as Prenatal screening, Newborn screening, and Reproduction Issues.  Dr. Klein predicted that the first areas that whole genome (clinical) sequencing would show clinical utility would be in studies of copy number, neuroblastomas and translocations. Robert said that NGS will likely democratize genomic medicine.

Several speakers echoed TGEN’s David Craig, Ph.D., Deputy Director for Bioinformatics and Professor of Neurogenomics,  comment that “the cost of NGS went up in 2011 because the analysis bottleneck is the culprit.”  My take on that is that in clinical NGS, the all-in $1,000 genome might be postponed to beyond 2014 by perhaps a few more years.

Ion Torrent Launches $1,000 Genome Sequencer in SF

On January 10, 2012, San Francisco, The Ruby Skye nightclub — just a block from the 30th Annual J.P.Morgan Healthcare Conference.

Life TechnologiesIon Torrent division hosted a reception and gave a product launch presentation for their new genome sequencer, The Ion Proton.  The invited guests enjoyed a cocktail reception for about 45 minutes before taking their seats at the theater-like presentation space.

Greg Lucier, Life’s CEO appeared, made some opening remarks and introduced Dr. Jonathan Rothberg, Ion Torrant’s founder.  Jonathan spoke for about thirty minutes and recapped the history of Sanger sequencing, 454 sequencing, comparisons to advancements in the computer industry and how his son got him to think about semiconductor-based sequencing.  He explained the rationale for using semiconductor technology for the Ion Torrent sequencer. Rothberg said that using the Ion Personal Genome Machine (PGM) to quickly sequence the genome of the virulent e.coli strain found in a contaminated food outbreak that killed 50 in Germany last year demonstrated the value of the PGM for public health. He said that as a result of the e.coli outbreak experience, Ion Torrent developed assays that turned out to be ideal for healthcare research applications.

At this point, Jonathan stepped back to a black draped structure at the back of the stage and unveiled a rack containing four new benchtop Ion Proton genome sequencers.  Rothberg spent about ten minutes explaining the key details about the new Ion Proton machine.  Life Technnologies is taking orders for the the Ion Proton, which sells for $149,000. He said that these machines are ideal for customers that need to sequence exomes and genomes.  The machines are designed to sequence exomes for $500 and genomes for $1,000 in 24 hours. The costs mentioned are material costs. The Ion Proton uses two new chips.  The Proton I chip has 10X the density of the current 318 chip (1.65 million wells) used in the PGM, is for exome sequencing and will be available later in the Spring. The Proton II chip has even higher density, with about 660 million wells, is for genome sequencing and will be available later in 2012.  Jonathan said that he was impressed that at a starting point of just using the PGM chemistry kit, the Ion Proton produced 200 base pairs of sequence.

Greg Lucier and Jonathan Rothberg on stage with Four Ion Protons

After the presentation, I had an opportunity to see and touch the new Ion Proton machine located at the back of the reception area and spoke with the Director of Product Marketing who was there to demo some of the new machine’s key features.  He said that Life Technologies was taking its sequencers on the road in their new Ion Torrent Bus which was parked out front.  After our discussion, I went outside and climbed aboard their mobile sequencing lab in a bus.  The two spokeswomen there said that bus the has two PGM machines and will soon be outfitted with two Ion Proton sequencers for a total of four machines that can sequence DNA as they drive.  I asked if they would be visiting the upcomimg genome conference at Marco Island.  They said yes and would also be visiting universities and commercial centers.

Pros and Cons of Sharing Personal Genomes on Social Media

I recently particpated in an online conference session held by NGS Leaders when CHI held its Beyond Sequencing conference in San Francisco recently on June 21, 2011. The session was moderated by NGSLeaders’ Eric Glazer and a panel of NGS experts.  The panelists included Kevin Davies, PhD.(Bio-IT World) Pillar Ossorio , PhD. (Univ. of Wisconsin), Johnathan Eisen, PhD, (UC Davis), and Kamamiesh (Kam) Patel, PhD. (Sandia National Labs).  This panel session was about “When People Share Their Genomes on Facebook.”  Both Kevin and Kam participated by telephone and the others were with a live audience in the meeting room.  The session used WebEx to link remote participants.

Kevin summarized the recent history of personal genomics services since the field was started by and deCode Genomics.  He pointed out that these services were marketed as services that enabled consumers to learn much about their genetic make up.  The services just provide information that might help people learn more about their risk for common disease or for geneology.  The customers’ genetic information is not to be used as a diagnostic. While the technology is initially based on microarrays and SNPs, some companies hoped to use NGS when the cost gets low enough.  However, Kevin said that the future of personal genomics is still in the hands of the FDA to decide on guidance to the industry.  Johnathan predicted that we will see a lot of push inside of genomes and personal microbiomes.  Microbiomes are the total kinds of microscopic things that live in or on our bodies.

As the discussion turned to social media, Kam said that the social media giants have big potential uses for health information.  A person could meet with their doctor, then the doctor could later access the person’s Facebook page and follow the progress of a treatment regimen.  But there are pros and cons to watch out for such as exploiters and cost issues.

Johnathan spoke about ‘crowd sourcing’ would be useful for health information.  He said that Facebook is only partly open.  He noticed a new push for open science such as to post lab notebooks on a social media site.  As for citizen science, he predicted that personal genomics, open science projects and citizen science will merge together.  When that happensm then anybody can make or use the information for their own open science projects.

A big question is who owns the personal genetic information held on social media sites.  Pillar, a lawyer, said that  we can think about this in terms of copyrights and patents.  She said that for example, when I get genetic information, I might just get a license for personal use, — it might be limited or unlimited.  The media site rules migth determine who can use the information, etc. At least we have the GINA law to protect our rights regarding employment and insurance discrimation.  However, she thinks that much of this is unknowns.  Pilliar said that the courts would likely say that if you put your genetic information on a public social media site, you are effectively giving away information to the public domain.  She said that people need to be educated about the subject so people would know who should participate.  She said that George Church has a screening process to weed out ignorant people.  He makes applicants take a genomics class and his program has a high cost.

Eric asked the panel when might clinical use will happen or become routine.  Johnathan said that technology will happen soon, but he did not know when clinical practice will happen.  Kevin said that the clinical use of personal genomics is happening now and cited the case of the boy from Wisconsin who was helped by NGS based diagnostics.  He said that there is a huge amount of genetic medical education that is needed.  Pillar said that she believes that the clinical context will take some time to work out.  My take on personal genomics and social media is that we are still in the early early days.

Low-Cost sequencers to Drive Growth in NGS Installed Base

First quarter announcements by two early makers of low-cost NGS machines suggests that brisk sales of the platforms will likely boost the overall installed base of NGS machines deployed into labs worldwide.  In mid-April, a spokes person from Roche 454 Life Scicences said that “We are pleased with the rapid adoption of the GS Junior System in the market.”  The person also said that 454 had “placed hundreds of GS Junior instruments in laboratories worldwide.”

To me, “hundreds of instruments” could be interpreted as at least 300-400 instruments. That’s quite a lot of 454 GS Junior instruments shipped since its launch last May. Most of what I have read about the GS Junior suggested that the instument have limited utility and may  have disappointed some users.  Brisk sales of the GS Junior is a surprise to me.

Life Technologies made an announcement about its low-cost Ion Torrent sequencer as part of its first quarter financial release. They said that their Q1 orders were greater than what they had expected.  They said the the strong order rate suggests that they might sell more Ion Torrent PGMs over the next 12-months that will exceed the installed base of the leading NGS instrument.  I assumed that he was referring to the installed base of the Illumina GA series of NGS machines. The Univ. of Birminkgham website shows that the self-reported installed base for Illumina GAs to be over 660.  I suspect that this website lags the real installed base by a few hundred. I read that LIfe Technologies had initial orders for 60 or so PGMs.  So I would expect that exceeding their original expections could be interpreted as 100 to 130 shipments for the PGMs for Q1. If LIfe’ shipment estimates do materialize, then sometime next April the installed base of Ion Torrent PGMs will reach about 670.

I estimate that the accumulated installed base of 454 GS Junior machines that might be deployed by next April would be about 650. So together, The installed base for the two low-cost NGS platforms might reach an installed base of 1,320 instruments.

If I assume that Illumina’s MiSeq instrument rolls out sometime in August and they have a run rate that is similar to the Ion Torrent, they might ship about 400 by next April.  Add that number to the mix and a conservative guestimate of the installed base for low-cost NGS machines might reach 1,720 machines by then. I can see that democratization of DNA sequencing will begin to take effect in mid-2012.

Next Gen. Sequencing for Dx – Exome v. Whole Genome?

While I was at CHI’s Molecular Medicine Tri-Con in San Francisco last week (Feb 23rd), I had a chance to sit in at a discussion table at the end of the day.  The topic at Table 6 was about diagnostic applications that used next generation sequencing (NGS).  About 16 people discussed the pros and cons of targeted resequencing versus whole genome sequencing. Karl Voelkerding M.D.,(Assoc. Professor, Pathology, Univ. of Utah; Medical Director, Advanced Technology and Bioinformatics, ARUP Laboratories), moderated the discussion. Karl said that NGS is being applied to multi-gene panels, exomes and whole genomes in clinical research and diagnostics. Each approach has different costs and complexity of data analysis and interpretation.

NGS for Multi-gene Panels v. Whole Genome
Karl started off by talking about multi-gene panels and NGS. Karl briefly talked about using multi-gene panels and Marfan Syndrome.  He said that the challenge involves sample preparation and noted that Fluidigm has a workable solution for this.

He asked the group “What’s being seen in Europe?” A person from Europe said that he has seen targeted NGS vs. whole genome NGS used by a fee-for-service company in Europe.  A person from Genomic Health said that, “if cost is not an issue, it’s OK to use whole genome.  But otherwise it’s better to use targeted resequencing.”  Karl said that at his lab, it takes over a year to do a CE- based multi-gene sequence [ vs. NGS].

Others at the table asked about costs.  The person from RainDance said that they have an in-solution capture method that could reduce costs.  Karl said that even there, there are non-trivial labor costs.  He said that “Some commercial companies do use robatic liquid handlers to reduce cost.”

Scenarios, Approaches, Costs
He said that this area is a moving target.  Amplified appproaches in multi-gene panels increase specificity for up to ten genes.  Otherwise if over ten genes, it takes many months of CE sequencing work. Researchers need to develop a special workflow for this type of CE- sequencing.  Karl said “An elusive goal is to make sequencing work like PCR.”  They are not there yet.

One person asked about simplifying the data content in a database by choosing some data as benign.  Karl said that academics are randomly updating their data by using a grad student or even an undergrad student.  But this approach gives inconsistant data quality.  He said that some commercial-based databases use more regularly scheduled updating.

He said that you need to ask the question “Are the genes associated with pathology?  Some genes are benign, some others are linked to disease.  We need to know, over time, what data items get classified as a changed data set.”
Some companies do targeted resequencing as a business and make IP from the database content. The database tells what is benign or what is something else.

A consultant asked “It would be interesting to see what in the database is predictive.”  Karl said “Extract the DNA, do PCR, do CE-seq, and analyze.”
The consultant also asked “What if you do NGS, then find genes, then pass data on to CE-seq to verify for Dx accuracy?” Karl said “Some research corelabs do exome sequencing for genome sequencing.  NHGRI is good with that approach.  He does 30x coverage at his lab.

Another person asked “What is the control level for false positives?
Karl said that, downstream, it depends on technologies used such as mass spec, v. NGS v. CE sequencing v. PCR.  Karl mentioned that the American College of Cardiology considered testing for hypertrophic cardiomyopathy (HCM)  and asked “Should we do multi-gene testing”  They test by using using echocardiograms.  Karl give the statitics for WW incidence.

So with the exome v. whole genome question. Karl asked, “When can you use gDNA for Illumina. The workflow is to do DNA sonograph, do Agilent Bioanalyzer 2100 to get total DNA, do qPCR to get fragment library which can go to the SOLiD or to the Illumina cluster [for HiSeq2000].

The sequencing workflow is:

  • Day 1 do gDNA
  • Day 2 do qPCR,  then transfer to Cbot
  • Day 3 run the HiSeq2000 at 2×100 for 8 days
  • Then run SeqTest, run QSeqTest, then output in Qfile format

Karl said it takes 105 days from start to end.

He said that, if you do exome sequencing, you need to do a purification step at the beginning, which adds 3-4 days to the workflow, but the exome sequencing is at a lower cost. Karl said the his lab is hooked up to the Univ. of Utah’s cluster computer and can do a data alignment in 1-day.  The cluster computer at the Univ. of Utah is also HIPPA compliant for privacy.

So cost drives exome sequencing. Karl said that “When doing exome sequencing you are doing a lot less sequencing, but you do more sample preparation.  You sequence on 2 lanes v. on 8 lanes [on Illumina].

Some List Prices
Karl gave some cost numbers.

  • For whole genome sequencing it costs $10K  with all reagents, including for library preparation.
  • For exome sequencing, it costs $1,200-$1,300 at 200X to 900X coverage.

So an answer for supporting multi-gene sequencing is to use exome sequencing of all genes in a panel.  e.g. Broad can sequence 2000 exomes per week. They streamlined a special workflow for this. Anyway, at the end of the day, you need to do down stream validation.

Consent Approaches that Should be Considered
A woman asked, “But in the clinical environment, what if you find other genetic information?, Some other genetic information?, Do you not tell the clinician?”
Karl said that “the key is informed consent.”  He said “ARUP is developing a tiered consent process — its mostly used for pediatrics now. So if they set out looking for one genetic area, but what if they find something else?  They age-level at age-14 for consent.”

Karl gave an example about the rare disease area at the NIH..  The NIH does exome sequencing.  Their success rate is 20% to identify a suspicious gene.  “So why just 20% with de novo mutations?”  He said that they are using exome sequencing and they just use a small population.  He mentioned a paper in Nature Genetics involving a group in the Netherlands  that saw a lot of power in NGS of a child that is an alternative to use laborious CE sequencing.

Karl said that the items not covered in the consented area are marked off.  He said that this is usually done in laboratory medicine.  When it comes to a recessive gene, the answer is often guided by family history.  Therefore “consent with tiering” is the way to be able to manage what diagnostic information is delivered to clinicians.  Karl wrapped up the discussion by saying that “NGS is pushing the envelope!”

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