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