ngs - basic principles and sequencing platforms

Next-generation-sequencingBasic principles and sequencing platforms

WIV-ISP workshop 10/10/2014

Annelies Haegeman

Institute for Agricultural and Fisheries ResearchPlant Sciences Unit

www.ilvo.vlaanderen.be

Agriculture and Fisheries Policy Area

10/10/2014 What is next generation sequencing? 2

Presentation outline

• Sanger sequencing (1977 – present)

• Next generation sequencing (2005 – present)• 454

• Ion Torrent

• Illumina

• 3rd generation sequencing (2012 – present)• PacBio or SMRT sequencing

• Oxford Nanopore

• Sequencing @ ILVO

What is next generation sequencing? 3

NGS experiment setup

DNA sample NGS Instrument Data

Library Preparation

Sequencing Data Analysis

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• Ion Torrent

• Illumina


• Oxford Nanopore


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Sanger sequencing (1977)

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Sanger sequencing

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http://en.wikipedia.org/wiki/Sanger_sequencing


Applied Biosystems

• 1987 : First sequencer (ABI370)

• 1996 : First capillary sequencer (ABI 310)

• Present : ABI 3500

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• Ion Torrent

• Illumina


• Oxford Nanopore


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454 sequencing

• 2005

• DNA is fragmented, adapter ligated

and denatured

• Emulsion PCR

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Rothberg, J.M. & Leamon, J.H. Nature Biotechnology 26, 1117 - 1124


454 sequencing

• Put beads and reagents in picotiter plate (one DNA bead

per well)

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454 sequencing

• Sequencing

• One bead = one read

• Reads up to 700 bp

• 1 million reads / run

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454 sequencing

GS Junior• ~ 400 bp reads

• Up to 100.000 reads / run

• 10h run time

GS FLX Titanium XL+• ~ 700 bp reads

• Up to 1 million reads/run

• 23h run time

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454 sequencing

• Pros

• First commercially available NGS technique

• Relatively long reads

• Relatively short run time

• Cons

• Accuracy problems with homopolymeric repeats

• Emulsion PCR

• Relatively few reads

• Relatively expensive

• R.I.P. 2016

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• Ion Torrent

• Illumina


• Oxford Nanopore


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• 2010, Life Technologies

• The first steps are similar to 454 sequencing

• Bind DNA to beads

• Emulsion PCR

• Chip with ion sensor


Ion Torrent sequencing

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• Semiconductor sequencing

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• Semiconductor sequencing on a chip (no imaging)

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Ion PGM• 3 types of chips

• 200 or 400 bp reads

• Up to 5.5 million reads / Ion 318 chip

• 4 – 7 h run time

Ion Proton• Up to 200 bp reads

• Up to 60-80 million reads

• 2 – 4 h run time

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• Pros

• Fast

• Relatively cheap

• Scalable (different chips)

• Cons

• Relatively high error rate

• Emulsion PCR

• Ion isothermal amplification chemistry (November 2014)

• isothermal amplification instead of emulsion PCR

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• Ion Torrent

• Illumina


• Oxford Nanopore


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Illumina sequencing

• 2006 (Solexa)

• Most popular sequencing technique nowadays!

0

2000

4000

6000

8000

10000

12000

454 solid ion

torrent

illumina pacbio

# studies in NCBI SRA database (01/2014)

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Illumina sequencing

Metzker, Nature Reviews Genetics, 2010, 11, 31-46


Paired end sequencing

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©Illumina


Illumina sequencing

MiSeq• Up to 2 x 300 bp reads

• 15 million single reads

• 2.7 days run time

HiSeq2500• Up to 2 x 125 bp reads

• 2 billion single reads

• 6 days run time

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Illumina sequencing

NextSeq 500• Up to 2 x 150bp reads

• Up to 400 million reads

• 1.2 days run time

HiSeq X Ten• 2 x 150 bp reads

• Up to 30 billion reads

• 3 days run time

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Illumina sequencing

• Pros

• Very high throughput

• Best price/bp

• Cons

• Relatively long run time

• Sequence quality decreases towards the end

• Imaging interference in low diversity libraries

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Advantages of NGS vs Sanger

• Cheap (cost per bp)• 1 bacterial genome (100x, 650Mb) < a 96-well plate (80 kb)

• (Very) high throughput (parallel sequencing)

• Multiplexing

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©Illumina


How do I choose a platform?

• Read length vs. number of reads (read depth)

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Overview

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Overview

Platform ChemistryRead

LengthRun Time Reads/Run Advantage Disadvantage

454 GS JuniorPyro-

sequencing400 8 h 0.1 M

Long read

length

High error rate,

expensive

454 GS FLX+Pyro-

sequencing700 23 h 1 M

Long read

length

High error rate,

expensive

Ion PGMProton

Detection200-400 4-7 h 5.5 M Short run time

High error rate, but

will improve

Ion ProtonProton

Detection200 2-4 h 80 M Short run time

High error rate, but

will improve

Illumina MiSeqBridge

amplificationup to 2x300 2.7 days 25 M

High-

throughput /

cost

Long run time

Illumina HiSeqBridge

amplification2x125 6 days 2000 M

High-

throughput /

cost

Short reads, long run

time

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• Illumina

• Ion Torrent


• Oxford Nanopore


PCR based

Single molecule

2nd

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PacBio sequencing

• 2011, Pacific Biosciences

• Single Molecule Real Time Sequencing (SMRT)

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PacBio sequencing

PacBio RSII• Up to 30 kb reads

• 50.000 reads

• Up to 3h run time

Often used in combination

with Illumina sequencing

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PacBio sequencing

• Pros

• No PCR

• Very long reads

• No problems with high GC content

• Can detect nucleotide modifications (e.g. methylation)

• Cons

• High error rate, but expected to improve

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• Illumina

• Ion Torrent


• Oxford Nanopore


PCR based

Single molecule

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Oxford nanopore sequencing

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Oxford nanopore sequencing

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• MinION portable sequencer

• Currently a beta version is being

tested in 500 selected labs

• Accuracy: 60-85%

• First reports on read lengths:

up to 79000 bases!

• Under development

• GridION

• PromethION





• Ion Torrent

• Illumina


• Oxford Nanopore


AnimalSciences

Cattle, Pig andSmall AnimalHusbandry

Fisheries andAquatic

Production

Marine Environment and Quality

Plant Sciences

CropProtection

Growth andDevelopment

Applied Genetics and

Breeding

CropHusbandry

Technology andFood Science

Agricultural Engineering

Product Quality andInnovation

Food Safety

Social Sciences

Agricultural and Farm

Development

RuralDevelopment

ILVO

What is next generation sequencing? 3910/10/2014

Plant Sciences

CropProtection

Growth andDevelopment

Applied Genetics and

Breeding

CropHusbandry

Sequencing @ ILVO


Whole genome shotgun (WGS) sequencing

Plant pathogens, mainly bacteria

Amplicon sequencing (16S rRNA)

Bacterial populations in soil

Transcriptome sequencing

Azalea, perennial ryegrass

Targeted resequencing

Perennial ryegrass

Genotyping-by-sequencing (GBS)

Perennial ryegrass, Phytophthora oomycetes

Sequencing @ ILVO


AnimalSciences

Cattle, Pig andSmall AnimalHusbandry

Marine Environment and Quality

Fisheries and

Aquatic

Production

Amplicon sequencing (16S rRNA)

Bacterial populations in:

- Cow rumen

- Marine environments (water, sediment, litter)

Whole genome shotgun sequencing(metagenomics)

- Pig gut (antibiotics resistance genes)

- Marine environments (plastic litter)

Genotyping-By-Sequencing (GBS)

Comb jelly


Sequencing @ ILVO

www.ilvogenomics.be

10/10/2014

Questions?

WIV-ISP workshop 10/10/2014

Institute for Agricultural and Fisheries ResearchPlant Sciences Unit

www.ilvo.vlaanderen.be

Agriculture and Fisheries Policy Area

[email protected]