Next-Generation Sequencing: an overview of technologies and applications

July 2014

Ken McGrathAustralian Genome Research Facility

Next-Gen Sequencing Edition

• Current rulers of the “throne”

• Sequencing by synthesis

• Each cycle extends and reads a single base

• Reads of up to 2x300bp

DNA(0.1-1.0 ug)

Sample preparation Cluster growth

5’

5’3’

G

T

C

A

G

T

C

A

G

T

C

A

C

A

G

TC

A

T

C

A

C

C

TAG

CG

TA

GT

1 2 3 7 8 94 5 6

Image acquisition Base calling

T G C T A C G A T …

Sequencing

Illumina Sequencing TechnologyRobust Reversible Terminator Chemistry Foundation

MiSeq

Illumina

NextSeq500HiSeq2500

MiSeq

Illumina

NextSeq500HiSeq2500

GAIIx

Illumina X Ten

ILLUMINA SEQUENCING SYSTEMS

•150 bp paired end reads ~120Gbp / run (~1 day)

NextSeq500•15

0 bp paired end reads ~ 180 Gbp/ run (2 days)

Illumina HiSeq 2500 Rapid SBS

•125 bp paired end reads ~ 1000 Gbp/ run (6 day)

Illumina HiSeq 2500 v4 SBS

•300 bp paired end reads ~15 Gb/run (2.3 days)

MiSeq v3

• 150bp paired end reads ~1800 Gb/run (3 days)HiSeq X Ten

ILLUMINA SEQUENCING SYSTEMS

•10 -15 million pass filter clusters per run

MiSeq v2•50

bp single reads (0.5 – 0.75 Gb/run)

~6hrs

•≥ 90% bases higher than Q30 at 50 bp

50 cycles

•150 bp paired end reads (3.0 – 4.5 Gb/run)

~24 hrs

•≥ 80% bases higher than Q30 at 2x150 bp

300 cycles

•2x250 bp paired end reads (5.0 - 7.5 Gb/run)

~40 hrs

•≥75% bases higher than Q30 at 2 x 250 bp

500 cycles

•20-25 million pass filter clusters per run

MiSeq v3

•2x 75 bp paired end reads (3.0 – 2.5 Gb/run)

~20 hrs

•≥ 85% bases higher than Q30 at 2 x 75 bp

150 cycles

•2x300 bp paired end reads (12.0 – 15.0 Gb/run)

~55 hrs

•≥ 70 % bases higher than Q30 at 2 x 300 bp

600 cycles

Illumina Summary Strengths Weaknesses

Lots of data Too much data

Low error rates Slower run times

Great choice of platform sizes Shorter reads

Paired-end reads

Pretty awesome Slept with brother

• Competing with illumina for market share

• Two technologies (sequencing by ligation, and semiconductor sequencing)

• Reads of up to 400bp

Ion Torrent

• Ion Semiconductor Sequencing

• Detection of hydrogen ions during the polymerization DNA

• Sequencing occurs in microwells with ion (pH) sensors

– No modified nucleotides

– No optics

Ion Torrent• DNA Ions Sequence

– Nucleotides flow sequentially over Ion semiconductor chip

– One sensor per well per sequencing reaction

– Direct detection of natural DNA extension– Millions of sequencing reactions per chip– Fast cycle time, real time detection

Sensor Plate

Silicon SubstrateDrain SourceBulk

dNTP

To column receiver

∆ pH

∆ Q

∆ V

Sensing Layer

H+

SOLiD

Life Technologies

Ion Torrent PGM Ion Torrent Proton

• 100 bp reads ~20 Gbp/run (Coming soon!)

Ion Torrent Chips

• 200bp and 400bp reads, 30-100Mb/run (1.5 hrs)314 Chip

• 200bp and 400bp reads, 300-1000 Mbp / run (2 hrs)316 Chip

• 200bp and 400bp reads, 600Mb-2Gbp / run (4.5 hrs)318 Chip

• 200 bp reads, 5-10 Gbp/run P1 Chip

P2 Chip

PG

MP

RO

TO

N

Life Technologies Summary Strengths Weaknesses

Fast run times Lower maximum data output

Scalable data outputs Read quality can vary

Longer reads (400bp)

Pretty Haven’t done much recently

• Current rulers of the throne

• Sequencing by synthesis

• Each cycle extends and reads a single base

• Reads up to 2x300bp

• Current rulers of the throne

• Sequencing by synthesis

• Each cycle extends and reads a single base

• Reads up to 2x300bp

• One of the first NGS platforms

• Pyrosequencing based

• Each cycle allows extension of a single base (A, C, G or T)

• Reads up to 800bp

454 Pyrosequencing

454 Pyrosequencing

454: Data Processing

Image Processing

Base-calling

Quality Filtering

SFF File

T Base Flow

A Base Flow

C Base Flow

G Base Flow

Raw Image Files

GS-FLX

Roche

FLX Jr

GS-FLX

Roche

FLX Jr

Roche

• Not over yet…

Stratos Genomics Genia Something else?

Roche Summary Strengths Weaknesses

Long reads (up to 800bp) High $ per base

Older technology

Platform soon unavailable

Had wolves Pretty much dead

• Competing with illumina for market share

• Two technologies (sequencing by ligation, and semiconductor sequencing)

• Reads of up to 400bp

• Competing with illumina for market share

• Two technologies (sequencing by ligation, and semiconductor sequencing)

• Reads of up to 400bp

• Single-molecule real-time sequencing (SMRT)

• Detection of individual bases as they extend (by light emission)

• Long Reads (up to 4x2.5kb)

PacBio

PacBio

• Higher error rates (~90%)

• Compensate by “looping” DNA to create multiple passes

PacBio

Zero-Mode Waveguides (ZMW)

PacBio Summary Strengths Weaknesses

Long reads (4x2.5kb) High $ per base

Single-molecule detection Higher error rate

Capable of Epigenetics Still to prove itself

Freakin’ Dragons! Keeps losing dragons

Oxford Nanopore

• Direct detection of individual bases as pass through a “nanopore”

• MinION and GridION

• No synthesis/extension

• Capable of VERY Long Reads (>100kb)

Oxford Nanopore Summary Strengths Weaknesses

Extra-Long reads (>100kb) Not yet available (alpha testing)

Single-molecule detection Very high error rates

Capable of Epigenetics Immature platform

Very cost effective

Exotic and powerful Steal babies

NGS Applications

• Whole genome sequencing (today)» De novo assembly» Structural variant detection» Comparative genomics

• RNAseq (later today)» Gene expression» Splice variants» Transcriptomics » MicroRNA

• Epigenomics (tomorrow)» Indirect (bisulphite)» Direct

• Targeted sequencing (Wed)» Hybrid capture» Amplicon resequencing

Data Quality

Read Length

Yield/Coverage

Hodor! (Thank You)