Next Generation
Sequencing in the
Clinical Laboratory
Norm Nelson, PhD
Senior Consultant
NCN Enterprises
Agenda
• 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
What is DNA Sequencing?
• 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.
The Human Genome Project
• 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
What is Next Generation
Sequencing (NGS)?
• 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
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
Potential applications of NGS
in clinical medicine
• Biomarker discovery
• Cancer mutation panels
• Mendelian disorders
• Bacterial ID and susceptibility
• Viral rare variant determination (HIV,
etc.)
• HLA tissue typing
• Mystery illnesses (medical odyssey)
Challenges that pose a threat to the
adoption of NGS in the clinical arena
• 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.
NCN Enterprises
Challenges to clinical adoption, cont’d
• 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 NGSbased 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
General strategic roadmap to
overcome these challenges
• 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
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
Places to find more information
about NGS
•
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:
Questions?