Next Generation Sequencing in the Clinical Laboratory

Norm Nelson, PhD

Senior Consultant

NCN 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

Illumina

HiSeq

2000

Illumina

MiSeq

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

Questions?