GS FLX+ System Delivering sequencing reads up to 1,000 bp · 2011-10-08 · GS FLX+ System Typical Performance Overview 11 Sequencing Kit New! XL+ XLR70 Read Lengths Up to 1,000 bp

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Xuemin Liu, PhD

GS FLX+ System

Delivering sequencing reads up to 1,000 bp

www.454.com

IMPORTANT NOTICE

Intended Use

Unless explicitly stated otherwise, all Roche Applied Science

and 454 Life Sciences products and services referenced in

this presentation/document are intended for the following use:

For Life Science Research Only.

Not for Use in Diagnostic Procedures.

Overview

GS FLX+ System Information

GS FLX+ Performance

GS FLX+ Application Data

GS Junior Introduction

454 Sequencing Read Length Evolution

Raising the bar in next-gen sequencing

GS 20

GS FLX Standard

100 bp

250 bp

2005 2007 2008

GS FLX Titanium

2011

GS FLX+ New!

Re

ad

Le

ng

th (

bp

)

1,000

500

250

750

GS Junior 400 bp

Up to

1,000 bp

www.454.com

GS Junior GS FLX+

Introducing the GS FLX+ System

Longer reads, more complete genome coverage

True capillary sequencing read lengths: Longest reads over 1 kb; greater

than 80% of total bases from reads longer than 500 bp

More complete genome coverage: Generate more accurate de novo

assemblies with fewer gaps and better coverage of repeat regions

Long GS FLX+ reads provide accurate scaffolds: Co-assemble with

short reads for more cost-effective de novo assemblies of complex genomes

More comprehensive transcript coverage: Single reads span more exons

for more complete transcript reconstruction

GS FLX+ System

Overview

Latest generation of the GS FLX System

Available as new instrument or on-site

upgrade to existing instrument

Choose from new GS FLX Titanium

Sequencing Kit XL+ for extra-long read

sequencing, or existing GS FLX Titanium

Sequencing Kit XLR70

Uses existing Rapid Library Prep and emPCR

kits, with slight protocol modifications for

extra-long read XL+ sequencing

New GS FLX+ Computing Station: Off-the-

shelf workstation optimized for XL+ long

read processing

7

Overview

System Information

Performance

Application Data

GS FLX+ Upgrade

How it works

New v2.6 instrument software:

- Sequencing cycles increased from

200 to 400 per run for XL+

Minor instrument modifications:

- New reagent door to accommodate

increased buffer volumes

- New chiller adapted for either XL+

or XLR70 reagent cassettes

- Waste ported into carboy outside

instrument for easy disposal

9

New Waste Disposal

Overview

System Information

Performance

Application Data

GS FLX+ System Typical Performance

Overview

11

Sequencing Kit New! XL+ XLR70

Read Lengths Up to 1,000 bp Up to 600 bp

Modal Read Length 700 bp 450 bp

Throughput Profile

- 85% of total bases from

reads >500 bp


reads >700 bp


reads > 300 bp


reads > 500 bp

Typical Throughput 700 Mb 450 Mb

Reads per Run ~1,000,000

Consensus Accuracy* 99.997% 99.995%

Run Time 23 hours 10 hours

Typical performance. Actual results depend on specific sample and genomic characteristics

*Consensus accuracy at 15x coverage E. coli.

GS FLX+ Read Length Distribution

Example Runs: Plant and animal

12

Budgie Bird (Parakeet)

Mode Read Length = 801 bp

Throughput = 860 Mb

Rice


Throughput = 753 Mb

GS FLX+ Read Length Distribution

Example Runs: Human BACs, bacteria

Human BACs


Throughput = 772 Mb

Bordetella pertussis


Throughput = 790 Mb

GS FLX+ Improvements

Significantly more bases from Sanger-like reads

- Example E. coli sequencing runs on the GS FLX+ System; one run using XLR70

backwards compatibility and one using XL+ sequencing kit

- New GS FLX+ generates significantly more bases from Sanger-like reads

(>500 bp in length)

0

1000000

2000000

3000000

0 100 200 300 400 500 600 700 800 900 1000

Ba

se

Co

un

t

Read Length (bp)

Base Count Distribution

GS FLX+ GS FLX

GS FLX + System

High accuracy over the long read

Source: Mapped E. coli reads from a GS FLX+ run and Illumina TruSeq run

Illumina TruSeq E. coli data set from http://www.illumina.com/truseq/tru_resources/datasets.ilmn

70%

75%

80%

85%

90%

95%

100%

0 100 200 300 400 500 600 700 800 900

Av

g.

Sin

gle

Re

ad

Ac

cu

rac

y

Read Length (bp)

GS FLX+

Illumina TruSeq

Overview

System Information

Performance

Application Data

GS FLX+ Applications

De novo Complex Genome Assembly

- Animal – Budgie Bird

- Assembly comparison using XL+ and XLR70

- Hybrid assembly using 454 Systems and

Illumina

cDNA/Transcriptome Assembly

Sequence Capture

Shotgun Metagenomics

17

Application Data

- De novo Genome Sequencing

- Transcriptome Sequencing

- Sequence Capture

- Shotgun Metagenomics


Transcriptome Sequencing

Sample: Drosophila larva; gene count -

16,600; 21,784 transcripts

Question: Do longer read lengths improve

transcriptome assembly?

Experimental Design:

- Random primed Drosophila larva cDNA

libraries (from mRNA)

- GS FLX cDNA Rapid library protocol

- Sequenced using XL+ and XLR70

Analysis: Compared mapping and assembly

metrics

Drosophila larva

Fragmentation

200 ng mRNA 50 to 2000 nt

AAAAAAAA

AAAAAAAA

First strand

cDNA synthesis

5’

3’

Fragmented mRNA

P-N6-primer

First strand cDNA

Second strand

synthesis

3’ 5’

5’

3’ 3’

5’ dscDNA with a 3’ overhang

End

repair

P-

A -

- A

-P

Blunt end / Phosphorylation and

the addition of overhanging A

Adaptor

ligation

Final Library

Enters the Rapid Library Prep Protocol

GS FLX Titanium

emPCR amplification

&

Sequencing

Adaptor Adaptor

cDNA Rapid Library Protocol Overview


Example Read Length Distribution Comparison

21

0

2000

4000

6000

8000

10000

12000

14000

0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

GS FLX+

GS FLX

Read Length

Re

ad

Co

un

t


Transcript Coverage Analysis

Transcripts are grouped according to their lengths:

- Small transcripts: ≤ 1.5 kb

- Medium transcripts: 1.5 to 4.5 kb

- Large transcripts: ≥ 4.5 kb

Within each and every transcript, every 5% of its length would be mapped

back to the reference and percentage of mappable hits are reported

Same number of reads from XLR70 and XL+ are compared

Transcript Coverage

Extra long reads provide much better coverage of long transcripts.

GS FLX+

GS FLX

Large Transcript Coverage

0

10

20

30

40

50

60

70

80

90

100

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

5' to 3' Transcript Coverage

%L

arg

e T

ran

sc

rip

ts (

>4

50

0)

wit

h E

ST

Ev

ide

nc

e

Small Transcript Coverage

0

10

20

30

40

50

60

70

80

90

100

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100


% S

ma

ll T

ras

nc

rip

ts (

<=

15

00

) w

ith

ES

T

Ev

ide

nc

e

Medium Transcript Coverage

0

10

20

30

40

50

60

70

80

90

100

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100


%M

ed

ium

Tra

ns

cri

pts

(>

15

00

&<

45

00

)

wit

h E

ST

Ev

ide

nc

e

≤ 1.5 kb 1.5 to 4.5 kb

≥ 4.5 kb

Mapping to the Chromosome GS FLX GS FLX+ Benefit

Number of Reads 1,557,790

% Reads Mapped 94% 92%

Number of Bases Mapped 547,105,393 732,429,546 +34%

Gene/Transcript/Exon Detection

% Genes 86% 86% -

% Transcripts 89% 89% -

% Exons 83% 85% +2%

% Avg. Transcript Length Covered 60% 74% +14%

% Chromosome Coverage

chr2L 24% 26% +2%

chr2R 29% 31% +2%

chr3L 25% 25% +2%

chr3R 26% 29% +3%

chr4 39% 40% +1%

chrX 25% 28% +3%


Mapping Results

Extra long reads improve transcriptome mappability

Transcriptome Sequencing Assembly Results – Decrease Ambiguity – Improve Assembly

Transcriptome Assembly

predicted gene locus

or transcriptional unit

predicted

transcripts

A set of reads assembled into different

combinations

The fewer isotigs and isogroups, the better de novo assembly of the

transcriptome.

GS FLX GS FLX+ Benefit

Number of Reads 1,557,790

Number of Isogroups 21323 19229 -10% (fewer is better)

Number of Isotigs 25019 24545 -2% (fewer is better)

Number of Bases 31,702,745 41,016,364 +29%

Average Isotig Size 1267 1671 +32%

N50 Isotig Size 1922 2565 +33%

Largest Isotig Size 26377 31533 +20%


Transcript Assembly Example

Reference TepII transcript is 4,535 bp in length

Assembled putative transcript from GS FLX+ EST data is an accurate

reconstruction of the full length reference transcript, including the splice

junctions

Reference

GS FLX

GS FLX+

Reference Source: Flybase Version 5.18

Transcriptome de novo Assembly

GS FLX+ Advancements

Extra-long reads:

• Increase coverage of full-length transcripts, particularly transcripts with

length ≥ 4.5 kb

• Span more exons and thus provide more information on splicing patterns

and spice junctions

• Generate more accurate and complete assemblies by reducing ambiguity

• Increase the total bases or genes that can be mapped uniquely to the

genome

Application Data



- Sequence Capture



Sequence Capture

Genome Info: Maize (plant), highly repetitive,

diploid. Known very complicated sequence.

Able to test assembly based upon exonic

capture by looking for intronic known bases

in the reads.

Questions: Can you capture increased GS

FLX+ fragment size? What is the value of

extra long reads?

Experimental Design: Library Prep

NimbleGen Array-based Capture XL+

Sequencing

Analysis: Reference mapping and

comparison to the capture design

NimbleGen Sequence Capture Workflow

Genomic DNA Library Preparation Hybridization Capture and Washing Amplification and

QC

Sequencing

Target

Regions

Amplify DNA and

Enrichment QC

Prepare with Next-

Gen Sequencing

Adaptors

Sequencing with

GS FLX+ System SeqCap EZ (Solution Capture)

Sequence Capture (Array Capture)

Array Probes

Sequencing

Reads

Sequence Capture

Results

Depth of

Coverage

GS FLX + reads span exons and complete intronic regions from one

exon to another.

Target Region

GS FLX+ Advancements

Sequence Capture

Extra-long reads:

• Span exons and complete intronic regions from one exon to another

• Improve mapability leading to better SNP/trait linkage

• Better resolution of homologs and paralogs

• Improve allele resolution of high ploidy genomes

Application Data



- Sequence Capture


Shotgun Metagenomics

Gut Microbes

Human discordant twins – obese

vs slim

Gut microbes isolated from both

and sequenced

To study if gut microbes play a

role in human obesity

Long reads improve identification

of microbes and provide more

species information

Confidential

GS FLX+ Sequencing Run Example

Metagenomics - Human Gut Microbes

GS FLX+ Demonstrated Applications

De novo Genome Assembly

- Resolve more repeats: Ideal for complex genome assemblies including animal, plant, and bacteria

- Improve assembly quality with fewer scaffolds/contigs at lower coverage


- Cover more exons / splice junctions with single reads

- Improve coverage of long transcripts

- Accurately reconstruct gene models

Sequence Capture

- Extend coverage beyond target regions into intergenic/intronic sequence

Metagenomics (Shotgun)

- Improve sensitivity and specificity of taxonomic assignment

GS FLX+ System

Summary

Longer capillary sequencing-like read lengths

Increased throughput

More complete genome and transcriptome coverage

Better results with improved economics!

Overview

GS FLX+ System Information

GS FLX+ Performance

GS FLX+ Application Data

GS Junior Introduction

GS Junior System

- An integrated solution from to sample to analysis

Day 3

数据分析

Day 2

测序

Day 1

样品制备

快速文库制备及emPCR制备

- 需要最少的实验室配备

• emPCR富集

• 即用的测序试剂

• 测序过夜 – 10小时运行时间

• 2 小时完成数据分析

• 界面友好的软件支持拼装，图谱分析以及扩增子变异分析

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Performance Summary

GS Junior System

GS Junior System

Throughput 35 million bases shotgun, 24 million bases amplicon (approx.)

Avg. Read Length 400 bases (approx.)

HQ Reads per Run 100,000 shotgun, 70,000 amplicon (approx.)

Accuracy Q20 read length at 400 bases (99% accuracy at 400 bases)

Run Time 10 hours sequencing, 2 hours data processing

Sample Input Purified gDNA, amplicons, cDNA, depending on application

*Per run specifications is for shotgun libraries, and can vary based on the organism and genomic content.

Reference organism is E. coli.

GS Junior System

- High Quality Data and Reliable Performance

Num

ber

of

reads

Readlength (bases)

One GS Junior System run produces reads from 50-600 or

more in length

Average is in 330-400 base range

Most reads are in the 450-550 base range

Q20 read length of 400 bases (99% accuracy at 400 bases)

GS Junior Instrument

GS Junior Titanium Kits GS Junior Attendant PC

GS Junior System in Your Lab

Next-gen sequencing & analysis in a corner of

your lab

7 ft 8 ft

Sequencing Based Assays

Full 454 Product Co-Development

Released HLA LSR ADME - RainDance

Future

TET2/CBL/KRAS (2011)

(Cancer/Leukemia)

Cancer Exons – NimbleGen

RUNX1 (2011)

(Cancer)

CFTR - Multiplicom

HLA Registry (2012)

(Bone Marrow Transfer)

HIV resistance CE (2012)

HCV resistance CE (2012)

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Beltsville, Maryland

New York, New York

Atlanta, Georgia

Where In the World is GS Junior?

In hundreds of labs worldwide!

Houston, Texas

Denver, Colorado

La Jolla, California

Corvallis, Oregon

Queensland, Australia

Perth, Australia

Ontario, Canada

Wenzhou, China

Copenhagen, Denmark

Oulu, Finland

Graz, Austria

Marcy L’etoile, France

Munich, Germany

Seoul, Korea

Tokyo, Japan

Auckland,

New Zealand

Barcelona, Spain

London, England

Sao Paulo, Brazil

Cape Town, South Africa

Taipei, Taiwan

Quezon City, Philippines

Riyadh, Saudi Arabia

Osaka, Japan

Warsaw, Poland

Tallinn, Estonia

Mountain View, Cali

Branford, Connecticut

Nashville, Tennessee

Geneva, Switzerland

Hong Kong, China

Note: Placements shown are a representative sample.

Disclaimer & Trademarks

For life science research only. Not for use in diagnostic procedures.

454, 454 SEQUENCING, 454 LIFE SCIENCE, EMCPR, GS FLX,

GS FLX TITANIUM, GS JUNIOR, NEWBLER, NIMBLEGEN, and

SEQCAP are trademarks of Roche.

Other brands or product names are trademarks of their respective holders.

GS FLX+ System Delivering sequencing reads up to 1,000 bp · 2011-10-08 · GS FLX+ System Typical Performance Overview 11 Sequencing Kit New! XL+ XLR70 Read Lengths Up to 1,000 bp

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