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

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

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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

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DepthOfCoverage

Genetics for Dummies 2017 NGS I - History and Technologies

Robert Kraaij Department of Internal Medicine r.kraaij@erasmusmc.nl

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Things to be addressed

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

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What will NGS bring us?

RFLP TaqMan Array Array and Imputation Regional Sequencing Full Genome Sequencing

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Overview

  • First Generation: Sanger sequencing
  • Next (Second) Generation
  • Third Generation
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1953: Double-Helix Model of DNA

James D. Watson and Francis Crick from wikipedia.org

 4 nucleotides  2 strands  A-T and C-G pairing

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1970: HindII the First Restriction Enzyme

Hamilton O. Smith

  • T T C G A A -

3’-

  • 5’
  • A A G C T T - -3’

5’-

from wikipedia.org

 isolation of clonal DNA fragments

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1977: Maxam & Gilbert Sequencing

Walter Gilbert from wikipedia.org

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Maxam & Gilbert Sequencing

G G+A C+T C

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1977: Sanger Sequencing

Frederick Sanger from wikipedia.org

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Sanger Sequencing

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Sanger Sequencing

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Sanger Sequencing

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G C A T

Sanger Sequencing

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G A T C

Sanger Sequencing

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Sanger sequencing landmarks

from wikipedia.org

  • 1977

bacteriophage φX174 5.4 kb

  • 1984

Epstein-Barr virus 170 kb

  • 1995

Haemophilus influenzae 1.8 Mb

  • 2001

Human 3 Gb

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June 26th, 2000: working draft, 95% gesequenced April 14th, 2003: finished: 99% gesequenced. Costs: $ 2.7 billion (instead of $ 3 billion) Timing: 1990 - 2003 (instead of 2005)

Bill Clinton Tony Blair Craig Venter Francis Collins

The Human Genome Project

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Overview

  • First Generation: Sanger sequencing
  • Next (Second) Generation
  • Third Generation
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Next Generation: Roche 454

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Roche 454

  • fragment DNA
  • clonal amplification
  • n bead by emPCR
  • load beads in

PicoTiterPlate

  • sequencing-by-

synthesis

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Roche 454

  • fragment DNA
  • clonal amplification
  • n bead by emPCR
  • load beads in

PicoTiterPlate

  • sequencing-by-

synthesis

micro-reactors water-in-oil emulsion

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Roche 454

  • fragment DNA
  • clonal amplification
  • n bead by emPCR
  • load beads in

PicoTiterPlate

  • sequencing-by-

synthesis

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Roche 454

  • fragment DNA
  • clonal amplification
  • n bead by emPCR
  • load beads in

PicoTiterPlate

  • sequencing-by-

synthesis

A

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Roche 454

  • fragment DNA
  • clonal amplification
  • n bead by emPCR
  • load beads in

PicoTiterPlate

  • sequencing-by-

synthesis

A  G  C  T  etc.

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Roche 454

  • fragment DNA
  • clonal amplification
  • n bead by emPCR
  • load beads in

PicoTiterPlate

  • sequencing-by-

synthesis

A A A T C G G G G G C A

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Next Generation: Ion Torrent

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Ion Torrent

  • fragment DNA
  • clonal amplification
  • n bead by emPCR
  • load beads on chip
  • sequencing-by-

synthesis

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Ion Torrent

  • fragment DNA
  • clonal amplification
  • n bead by emPCR
  • load beads on chip
  • sequencing-by-

synthesis

A

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Ion Torrent

  • fragment DNA
  • clonal amplification
  • n bead by emPCR
  • load beads on chip
  • sequencing-by-

synthesis

T  G  A  C  etc.

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Ion Torrent

  • fragment DNA
  • clonal amplification
  • n bead by emPCR
  • load beads on chip
  • sequencing-by-

synthesis

A A A T C G G G G G C A

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Next Generation: Illumina

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Sequencing Workflow

DNA isolation Library preparation Sequencing Data analysis

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Sequencing Workflow

DNA isolation Library preparation Sequencing Data analysis

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Sequencing Workflow

DNA isolation Library preparation Sequencing Data analysis

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Illumina sequencing

  • fragment DNA
  • clonal amplification
  • n flowcell by bridgePCR
  • sequencing-by-synthesis
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Bridge amplification

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Illumina sequencing

  • fragment DNA
  • clonal amplification
  • n flowcell by bridgePCR
  • sequencing-by-synthesis
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Sequencing by synthesis

HP1 primer anneals to adapter

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Sequencing by synthesis

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A + C + T + G

Sequencing by synthesis

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

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Sequencing by synthesis

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Per Cycle Imaging

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G A T C

Per Cycle Imaging

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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 50k$ 100k€ 250k€ 700k$ 4250 $/WG 3500 $/WG

Illumina: Normal flow cell technology

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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 900k$ 5 x 1.2M$ 10 x 1M€ 1M€ 2500 $/WG 1500 $/WG 1000 $/WG 1200 $/WG

Illumina: Patterned flow cell technology

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Illumina: Patterned flow cell technology

  • Patterned flowcell
  • Billions of nanowells
  • Extreme high density
  • No overlapping clusters
  • Special polymerase?
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Illumina Whole Genome Sequencing

price per whole genome ($) 5,000 - 10,000 - 0 - price per system

MiSeq 10,000$ NextSeq 4,250$ HiSeq2500 3,500$ HiSeq3000/4000 2,500$ HiSeqX Five 1,500$ HiSeqX Ten 1,000$

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Overview

  • First Generation: Sanger sequencing
  • Next (Second) Generation
  • Third Generation
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Third generation sequencing = single molecule sequencing

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Third Generation: PacBio

RS Sequal

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  • no DNA amplification
  • real-time imaging of

DNA polymerase

  • sequencing-by-

synthesis

PacBio

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SMRT technology

  • Library prep
  • Circular DNA
  • SMRT cell
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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

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Third Generation: Oxford Nanopore

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Oxford Nanopore

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Oxford Nanopore

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Oxford Nanopore

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

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

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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