NGS I - History and Technologies Robert Kraaij Department of - PowerPoint PPT Presentation

DepthOfCoverage Genetics for Dummies 2017 NGS I - History and Technologies Robert Kraaij Department of Internal Medicine r.kraaij@erasmusmc.nl

Things to be addressed Sanger sequencing: how it began NGS: many short reads that might contain errors Third generation sequencing: now available!

What will NGS bring us? RFLP TaqMan Array Array and Imputation Regional Sequencing Full Genome Sequencing

Overview • First Generation: Sanger sequencing • Next (Second) Generation • Third Generation

1953: Double-Helix Model of DNA James D. Watson and Francis Crick  4 nucleotides  2 strands  A-T and C-G pairing from wikipedia.org

1970: HindII the First Restriction Enzyme - A A G C T T - - 3’ 5’ - - T T C G A A - 3’ - - 5’ Hamilton O. Smith  isolation of clonal DNA fragments from wikipedia.org

1977: Maxam & Gilbert Sequencing Walter Gilbert from wikipedia.org

Maxam & Gilbert Sequencing G G+A C+T C

1977: Sanger Sequencing Frederick Sanger from wikipedia.org

Sanger Sequencing

Sanger Sequencing

Sanger Sequencing

Sanger Sequencing G C A T

Sanger Sequencing G A T C

Sanger sequencing landmarks • 1977 bacteriophage φ X174 5.4 kb • 1984 Epstein-Barr virus 170 kb • 1995 Haemophilus influenzae 1.8 Mb • 2001 Human 3 Gb from wikipedia.org

The Human Genome Project Bill Clinton Tony Blair Craig Venter Francis Collins June 26 th , 2000 : working draft, 95% gesequenced April 14 th , 2003 : finished: 99% gesequenced. Costs : $ 2.7 billion (instead of $ 3 billion) Timing : 1990 - 2003 (instead of 2005)

Overview • First Generation: Sanger sequencing • Next (Second) Generation • Third Generation

Next Generation: Roche 454

Roche 454 • fragment DNA • clonal amplification on bead by emPCR • load beads in PicoTiterPlate • sequencing-by- synthesis

Roche 454 • fragment DNA • clonal amplification on bead by emPCR • load beads in micro-reactors PicoTiterPlate water-in-oil emulsion • sequencing-by- synthesis

Roche 454 • fragment DNA • clonal amplification on bead by emPCR • load beads in PicoTiterPlate • sequencing-by- synthesis

Roche 454 A • fragment DNA • clonal amplification on bead by emPCR • load beads in PicoTiterPlate • sequencing-by- synthesis

Roche 454 • fragment DNA A  G  C  T  etc. • clonal amplification on bead by emPCR • load beads in PicoTiterPlate • sequencing-by- synthesis

Roche 454 • fragment DNA • clonal amplification on bead by emPCR • load beads in PicoTiterPlate • sequencing-by- synthesis A A A T C G G G G G C A

Next Generation: Ion Torrent

Ion Torrent • fragment DNA • clonal amplification on bead by emPCR • load beads on chip • sequencing-by- synthesis

Ion Torrent A • fragment DNA • clonal amplification on bead by emPCR • load beads on chip • sequencing-by- synthesis

Ion Torrent • fragment DNA T  G  A  C  etc. • clonal amplification on bead by emPCR • load beads on chip • sequencing-by- synthesis

Ion Torrent • fragment DNA • clonal amplification on bead by emPCR • load beads on chip • sequencing-by- synthesis A A A T C G G G G G C A

Next Generation: Illumina

Sequencing Workflow Library Data DNA preparation Sequencing analysis isolation

Sequencing Workflow Library Data DNA preparation Sequencing analysis isolation

Sequencing Workflow Library Data DNA preparation Sequencing analysis isolation

Illumina sequencing • fragment DNA • clonal amplification on flowcell by bridgePCR • sequencing-by-synthesis

Bridge amplification

Illumina sequencing • fragment DNA • clonal amplification on flowcell by bridgePCR • sequencing-by-synthesis

Sequencing by synthesis HP1 primer anneals to adapter

Sequencing by synthesis

Sequencing by synthesis A + C + T + G

Sequencing by synthesis A A A T C G G G G G C A

Sequencing by synthesis

Per Cycle Imaging

Per Cycle Imaging G A T C

Illumina: Normal flow cell technology MiniSeq MiSeq NextSeq500 HiSeq2500 2 x 150 b 2 x 300 b 2 x 150 b 2 x 125 b 6.6 Gb 13 Gb 100 Gb 450/900 Gb 22M clusters 22M clusters 0.4B clusters 2B/4B clusters 1 day 3 days 1 day 6 days 100k € 250k € 50k$ 700k$ 4250 $/WG 3500 $/WG

Illumina: Patterned flow cell technology HiSeq4000 HiSeqX Five HiSeqX Ten NovaSeq6000 2 x 150 b 2 x 150 b 2 x 150 b 2 x 150 b 0.65/1.3 Tb 0.8/1.6 Tb 0.8/1.6 Tb 0.85/1.7 Tb 2/4 B clusters 2.5/5 B clusters 2.5/5 B clusters 2.8/5.6 B clusters 4 days 3 days 3 days 2 days 10 x 1M € 1M € 900k$ 5 x 1.2M$ 2500 $/WG 1500 $/WG 1000 $/WG 1200 $/WG

Illumina: Patterned flow cell technology  Patterned flowcell  Billions of nanowells  Extreme high density  No overlapping clusters  Special polymerase?

Illumina Whole Genome Sequencing 10,000 - price per whole genome ($) MiSeq 10,000$ 5,000 - NextSeq 4,250$ HiSeq2500 HiSeq3000/4000 3,500$ 2,500$ 0 - HiSeqX Five 1,500$ HiSeqX Ten 1,000$ price per system

Overview • First Generation: Sanger sequencing • Next (Second) Generation • Third Generation

Third generation sequencing = single molecule sequencing

Third Generation: PacBio RS Sequal

PacBio • no DNA amplification • real-time imaging of DNA polymerase • sequencing-by- synthesis

SMRT technology  Library prep  Circular DNA  SMRT cell

SMRT technology  >10kb reads  1 Gb output  Better chemistry  De novo assembly  Haplotyping  Variant calling Posted February 10, 2014 The Genomics Resource Center University of Maryland http://www.igs.umaryland.edu

Third Generation: Oxford Nanopore

Oxford Nanopore

Oxford Nanopore

Oxford Nanopore  Library prep  1D or 2D reading  >100kb reads  Not many reads

Oxford Nanopore  6 bases in pore  6x base calling  Caller development  Community  Not ready yet  “ Illumina in 2007 ”  Big improvement 2017

Oxford Nanopore

Things to Remember Next Generation Sequencing techniques will allow to interrogate every single base in a genome Sanger Sequencing is the first generation of sequencing which is based on chain termination emulsionPCR is a PCR technique that allows to perform millions of PCR reactions in one tube bridgePCR: ditto on a flowcell NGS: many short reads that contain errors