Making Sense of NextGen Sequencing Presented by Kelly Gonzalez, MS, CGC Senior Manager of Clinical Genomics

Overview • Next Generation Sequencing (NGS): The Basics – How does the technology work? – Terminology – NGS applications • NGS panels vs whole genome/whole exome

• NGS Clinical Applications • Future & Emerging Uses of NGS

Slide created by Kelly Gonzalez, MS, CGC, Ambry Genetics

Cost of Sequencing Over Time THEN: Human Genome Project: $3 billion and 13 years NOW: Sequencing centers and laboratories: ~$15K and ~15 days

Data from the National Human Genome Research Institute (NHGRI)

Slide created by Kelly Gonzalez, MS, CGC, Ambry Genetics

Traditional vs. NextGen Sequencing SANGER SEQUENCING: 1 SEQUENCE READ PER BP

NGS: MULTIPLE SEQUENCE READS PER BP

Slide created by Kelly Gonzalez, MS, CGC, Ambry Genetics

Next-Gen Technologies on the Market »

Roche  454 (GS FLX Titanium, GS Junior)

»

Illumina – – – –

»

HiSeq (HiSeq2000, HiSeq 1000) Genome Analyzer (IIx, IIe) MiSeq iScanSQ

»

Pacific Biosciences  PacBio RS

»

Helicos  HeliScope*

*Recently stopped selling instruments in favor of a service provider approach

Life Technologies  SOLiD4  Ion Torrent  Starlight Slide created by Kelly Gonzalez, MS, CGC, Ambry Genetics

Generic Overview Of NGS 1. Library Preparation

2. Clonal Amplification

3. Sequencing

Voelkerding KV (2010) Next Generation Sequencing for Clinical Diagnostics-Principles and Application to Targeted Resequencing for Hypertrophic Cardiomyopathy. Journal of Molecular Diagnostics 5(12): 539-551

Slide created by Kelly Gonzalez, MS, CGC, Ambry Genetics

1. Library Preparation Input DNA fragmented  Shearing by: • nebulization • sonication • enzymatic digestion

Fragments have terminal overhangs  Blunt-end repair & phosphorylation Adapter ligation  Platform-specific adapters are ligated to the fragments Final Library  Short DNA fragments with platform-specific adapters

Voelkerding KV (2010) Next Generation Sequencing for Clinical Diagnostics-Principles and Application to Targeted Resequencing for Hypertrophic Cardiomyopathy. Journal of Molecular Diagnostics 5(12): 539-551

Slide created by Kelly Gonzalez, MS, CGC, Ambry Genetics

2. Clonal Amplification

Amplify the fragments  Emulsion PCR – oil in water bead-based – ONE BEAD = ONE FRAGMENT = ONE SEQUENCE READ  Bridge amplification – solid surface, flow-cell-based – ONE CLUSTER = ONE FRAGMENT = ONE SEQUNCE READ Voelkerding KV (2010) Next Generation Sequencing for Clinical Diagnostics-Principles and Application to Targeted Resequencing for Hypertrophic Cardiomyopathy. Journal of Molecular Diagnostics 5(12): 539-551

Slide created by Kelly Gonzalez, MS, CGC, Ambry Genetics

3. Sequencing

Pyrosequencing

» Sequential incorporation of nucleotides  luminescence

Sequencing by ligation

» Introduction of oligonucelotide probes  fluorescence

Reversible dye terminators »

Incorporation of reversible dye terminators  fluorescence

Voelkerding KV (2010) Next Generation Sequencing for Clinical Diagnostics-Principles and Application to Targeted Resequencing for Hypertrophic Cardiomyopathy. Journal of Molecular Diagnostics 5(12): 539-551

Slide created by Kelly Gonzalez, MS, CGC, Ambry Genetics

Terminology: Next-Gen What does Next-Gen really mean?

DNA-sequencing methods that involve chemical assays other than the traditional Sanger deoxy-chain-termination method.1 •

Next Generation (NextGen) Sequencing, AKA: – Deep Sequencing – Massively Parallel Sequencing – Second/Third-generation Sequencing • 2nd: Undergo amplification of the template molecules • 3rd: Single-molecule sequencing

1. Lupski, et al (2010) Whole Genome Sequencing in a Patient with Charcot-Marie Tooth Neuropathy. NEJM. 362(13):1181-1191.

Slide created by Kelly Gonzalez, MS, CGC, Ambry Genetics

Terminology: Reads, Read length Ex. Illumina’s sequencing by synthesis

# of sequencing cycles = Read length

C Reads

A

T

C

G

T

... C

C

C

..

Slide created by Kelly Gonzalez, MS, CGC, Ambry Genetics

Terminology: Read, Read length No generally accepted standards exist for read length, but the following guidelines apply1: – Short reads – sequences < 50 consecutive bases – Mid-length reads – sequences >51, but 400, but 20 years •

Molecular/Cytogenetic: – – – –

Karyotype Fragile X SNP chromosome microarray Mitochondrial DNA sequencing

– XLMR panels (>90 genes)

ALL RESULTS NORMAL



Biochemical: – – – – – – – – – – –

Amino acids, organic acids Lactate, pyruvate Lysosomal enzyme panel 7-dehydrocholesterol Very long chain fatty acids Plasmalogens, Pipecolate Uric acid, HPRT enzyme Urine GAGs and oligos Acylcarnitines Guanidinoacetate/creatinine CDG

Exome Sequencing Results

Exome Sequencing Results • ELP2 gene mutations recently identified in two consanguineous families with autosomal recessive non-syndromic intellectual disability

Exome Sequencing Results

Impact of Diagnosis • Determine inheritance pattern – Autosomal recessive, not X-linked

• Accurate recurrence risk counseling for sibs – Sister is carrier, healthy brother is not

• Psychosocial benefits – Relief of mother’s guilt, peace of mind

Paradigm Shift in Molecular Genetics Traditional Sequencing Laboratory Directors & Genetic Counselors

NextGen Sequencing

Laboratory Directors Genetic Counselors

Laboratory Technicians

Bioinformaticians Laboratory Technicians

Slide created by Kelly Gonzalez, MS, CGC, Ambry Genetics

Future of NGS • Non-invasive prenatal diagnosis for aneuploidies (*CURRENT USE) • Exome/genome sequencing in clinical trials for patients with end-stage cancer • NGS for pharmacogenomics

Thank you! Any questions? Slide created by Kelly Gonzalez, MS, CGC, Ambry Genetics