Next Generation Sequencing in the Clinical Laboratory

Norm Nelson, PhDSenior ConsultantNCN Enterprises

• What is Next Generation Sequencing (NGS)?

• What are potential applications of NGS in human health care?

• What are some obstacles to implementing NGS in the clinical laboratory?

• Places to find more information about NGS

Agenda

• From Wikipedia: DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It includes any method or technology that is used to determine the order of the four bases—adenine, guanine, cytosine, and thymine—in a strand of DNA.

What is DNA Sequencing?

• The human genome is the genetic blueprint of the human body

• The Human Genome Project was formally initiated in October, 1990, with the goal of sequencing the entire genome from one individual

• The project was completed in 2003

• The cost of generating the sequence for one genome was close to $1 billion

• The Sanger method was used to sequence the DNA

The Human Genome Project

• One human genome is not enough to even begin to understand the impact of this genetic information on health care. Thousands of sequences are desired…for starters.

• The Sanger method is too slow and costly to be practical to support generation of this many sequences

• New, “next generation” methods have been developed over the last 10 years that are absolutely breathtaking

What is Next Generation Sequencing (NGS)?

Illumina sequencing chemistry

3

IlluminaHiSeq 2000

IlluminaMiSeq

Ion Torrent

Roche 454

DNA Library Prep (~ 4.5 hours)

Emulsion PCR(~8 hours)

Sequencing(~ 10 hours)

Pacific Biosciences

Complete Genomics

Library Construction

Production of DNA Nano-balls (DNB)

Oxford Nanopore

IBM

Precipitous drop in costs

• Biomarker discovery

• Cancer mutation panels

• Mendelian disorders

• Bacterial ID and susceptibility

• Viral rare variant determination (HIV, etc.)

• HLA tissue typing

• Mystery illnesses (medical odyssey)

Potential applications of NGS in clinical medicine

• NGS technology is still rapidly evolving– By the time a selected approach is

commercialized for use as a diagnostic (product development, clinical trials, FDA approval, etc.) it may be obsolete.

– Full automation with FDA approval takes years and millions of dollars to develop. This could be a wasted investment if the technology changes and the assays can no longer run on the instrument.

– A completely new and novel approach to sequencing that completely changes the way commercialization is pursued could be just around the corner.

Challenges that pose a threat to the adoption of NGS in the clinical arena

NCN Enterprises

• Current workflows not optimal for the clinical laboratory– Too cumbersome, technically challenging & slow– Automation not complete– Prep from real clinical specimen typically not

rigorously addressed– Current workflows platform-specific– Expensive– Reagents that meet demanding performance

standards may be difficult to manufacture in bulk in an industrial setting

– Reagent stability may not be commercially viable in the diagnostics space

Challenges to clinical adoption, cont’d

• Data manipulation still too challenging– Significant IT infrastructure required

• This can be somewhat reduced by utilizing The Cloud

– Bioinformatics staff required– Query against relevant databases cumbersome

and not standardized

Challenges to clinical adoption, cont’d

• Interpretation of data problematic– The physician ordering a test just wants a

simple, actionable result they can use to make a treatment decision

• One current protocol for use of NGS data in cancer diagnostics, for example, is to convene a board of experts (oncology, genomics, bioinformatics, pathology, ethics, etc.) to discuss cases one by one and make treatment recommendations

– Clinically validated and adequately annotated databases against which to compare patient data are currently inadequate

– Biomarker sets with demonstrated clinical utility are sorely lacking

Challenges to clinical adoption, cont’d

• Regulatory hurdles– FDA approval for IVD assays is much more

rigorous than for LDTs, RUOs and even CE marked assays

– Regulatory guidelines for approval of NGS-based IVDs are unclear, as this part of the process is in its infancy

– NGS-based assays are inherently highly multiplex, which creates a huge unknown regarding the degree of validation required for FDA approval (will all possible targets require independent validation?)

– Clinical validity vs. clinical utility an issue

Challenges to clinical adoption, cont’d

• Intellectual property protection– The patentability of genetic biomarkers in

question• Landmark case (still in progress) – ACLU v. Myriad

Genetics and the University of Utah Research Foundation, which hold patents on the genes BRCA1 and BRCA2

– AKA Association for Molecular Pathology, et al. v. U.S. PTO– Argument – “products of nature”– 3/10 – A New York federal court ruled the patents invalid– 7/11 – Appeals court ruled genes patentable but not methods

to compare those genes– 3/12 – Supreme court vacated the appeals court decision and

instructed them to reconsider in light of Mayo v. Prometheus

– From a IVD Manufacturer’s perspective, potential lack of patent protection brings into question the value proposition of developing and acquiring FDA approval for NGS-based diagnostic assays

Challenges to clinical adoption, cont’d

• Reimbursement– Who will pay…and how much?– The issues of clinical utility and highly

multiplexed assays (many answers) raised earlier have huge impact on reimbursement decisions as well

– The assays will be expensive to develop, acquire approval and manufacture, and a cloudy reimbursement picture will once again bring into question the value proposition for an IVD manufacturer

– As with the regulatory piece, the reimbursement issues are in their infancy

Challenges to clinical adoption, cont’d

• Fund directed R&D efforts towards development of a fully automated clinical sample to sequencing-ready template workflow that is rapid, simple, inexpensive and manufacturable– Ideally should be easily adaptable for use

with different sequencing approaches– Should include automated chip loading

interface and ability to easily integrate with the sequencing piece

General strategic roadmap to overcome these challenges

• Engage in a wide variety of clinical collaborations– Biomarker discovery– Biomarker validation– Clinical utility demonstration– Access to clinical sample sets– These activities are extremely important in

regards to ultimate regulatory approval and reimbursement

General strategic roadmap to overcome these challenges

• Develop proprietary content beyond that which may or may not be patentable– Database– Software– Other…

These features may be key in developing a competitive advantage

General strategic roadmap to overcome these challenges

• Engage early and often in diverse forums and workshops created to discuss these challenges– National Cancer Institute (NCI)– Institute of Medicine (IOM)– Food and Drug Administration (FDA)– College of American Pathologists (CAP)– Association for Molecular Pathology (AMP)– National Human Genome Research Institute

(NHGRI)– American College of Medical Genetics and

Genomics (ACMG)Be part of the solution

General strategic roadmap to overcome these challenges

Is this any longer far fetched?

What about this:

• Rapid sample prep and template construction in a disposable microfluidic cartridge

• Sequencing in 15 minutes

• Data workup through a high speed wireless internet connection to a cloud computing center

• Sequence compared against a large and growing database

• Results sent directly to your PDA

• DNA sequencing - Wikipedia, the free encyclopedia

• Human Genome Project Information (http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml)

• All About The Human Genome Project (http://www.genome.gov/10001772)

• Instrument manufacturers (www.illumina.com/; www.iontorrent.com/; www.pacificbiosciences.com)

• General information about NGS (http://www.blueseq.com/; http://nextgenseek.com; http://seqanswers.com/forums/index.php)

• NGS for breast cancer recurrence risk (http://dx.plos.org/10.1371/journal.pone.0040092)

• Solution to mystery illness (http://www.illumina.com/documents/icommunity/article_2011_10_SkepticTurnedBeliever.pdf)

• Clinical Sequencing article:

Places to find more information about NGS

2012-10 Clin Dx Genome Seq; Schrijver, et al (J Mol Path).pdf

Questions?