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

from wikipedia.org

4 nucleotides

2 strands

A-T and C-G pairing

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

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

G C A T

Sanger Sequencing

G A T C

Sanger Sequencing

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

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

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

PicoTiterPlate

• sequencing-by-

synthesis

micro-reactors

water-in-oil

emulsion

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

PicoTiterPlate

• sequencing-by-

synthesis

A

Roche 454

• fragment DNA

• clonal amplification

on bead by emPCR

• load beads in

PicoTiterPlate

• sequencing-by-

synthesis

A G C T etc.

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

• fragment DNA

• clonal amplification

on bead by emPCR

• load beads on chip

• sequencing-by-

synthesis

A

Ion Torrent

• fragment DNA

• clonal amplification

on bead by emPCR

• load beads on chip

• sequencing-by-

synthesis

T G A C etc.

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

DNA

isolation

Library

preparation Sequencing

Data

analysis

Sequencing Workflow

DNA

isolation

Library

preparation Sequencing

Data

analysis

Sequencing Workflow

DNA

isolation

Library

preparation Sequencing

Data

analysis

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

A + C + T + G

Sequencing by synthesis

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

Sequencing by synthesis

Sequencing by synthesis

Per Cycle Imaging

G A T C

Per Cycle Imaging

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

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

Illumina: Patterned flow cell technology

Patterned flowcell

Billions of nanowells

Extreme high density

No overlapping clusters

Special polymerase?

Illumina Whole Genome Sequencing p

rice p

er

wh

ole

gen

om

e (

$)

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$

Overview

• First Generation: Sanger sequencing

• Next (Second) Generation

• Third Generation

Third generation sequencing =

single molecule sequencing

Third Generation: PacBio

RS

Sequal

• no DNA amplification

• real-time imaging of

DNA polymerase

• sequencing-by-

synthesis

PacBio

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