Semiconductor Sequencing for...

Ion TorrentIon TorrentSemiconductor Sequencing for Life™

1

• Ion Technology• Ion Workflow• Ion Performance &Data• Applications

Table of Contents

• Applications

3

Confidential and Proprietary—DO NOT DUPLICATE4

Ion Technology

Disruptive Technology

Main Frame Mini Computer Personal Computer

5

Generations are defined by who can use them.

Sanger Sequencing Next-Gen Sequencing Ion Semiconductor Sequencing

The Chip is the Machine™

Scalability

Simplicity

6

Speed

Simple Natural Chemistry

Eliminate source of sequencing errors:– Modified bases– Fluorescent bases– Laser detection– Enzymatic amplification cascades

Eliminate source of read length limitations:– Unnatural bases– Faulty synthesis– Faulty synthesis– Slow cycle time


Sequencing: Flows and Cycles

T A C G T A C G T

• A “flow” is the event of exposing the chip to one particular dNTP (T, A, C, or G), followed by a washing step

• A “cycle” is four consecutive dNTP flows: for instance, T-A-C-G = 1 cycle• Our flow order is a repeat of:• ‘TACGTACGTCTGAGCATCGATCGATGTACAGC’

C G A CG T AC GT A

Do Not Duplicate

Ion Sphere -----Primer------ A G T C A A G C G T C C C A T G

Sequence of InterestKey Sequence

Cycle 1 Cycle 3Cycle 2… etc

T

A

C

G

Cycle 1

...---Ion Sphere™Particle




T A C G T A C G T C G A CG T AC GT A

T

Do Not Duplicate



C


T

A

C

G

Cycle 2

...---Ion Sphere™Particle


T A C G T A C G T C G A CG T AC G



T A

T

Do Not Duplicate



C


T

C

G

T

Cycle 3+

A G...---Ion Sphere™

Particle

T

Fast Direct Detection

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

dNTP

∆ pH

∆ Q

H+


Sensor Plate

Silicon SubstrateDrain SourceBulk To column

receiver

∆ Q

∆ V

Sensing Layer

Precise Measurement of Each Incorporation

• Fast sequencingA few seconds per incorporation

• High signal to noiseMany data points per incorporation traceR

aw S

igna

l

Single Well Incorporation Trace

Many data points per incorporation trace

• Enables high raw accuracy


Raw

Sig

nal

Observations

Frames (15 frames = 1 second)

Wash WashNucFlow

Unprecedented Scalability…

1 Gb

10 Gb

100 Gb

2011 2012 2013

10 Mb

100 Mb

1 Gb

The content provided herein may relate to products that have notbeen officially released and is subject to change without notice.

Introducing the Ion Proton™ SequencerThe Benchtop Genome Center

21 The content provided herein may relate to products that have notbeen officially released and is subject to change without notice.


Ion Workflow

Technology Summary


Compatible with existing libraries *

Ion Workflow

DNA / RNAAA

CC

CompatibleLibrary Prep

BB

Storage of 1,000s of runs

Fast scalable DNA sequencing

Automated sample preparation

Sequencing1.5 hours

DD

Template Prep4 hours

CC

CompatibleFASTQ Data0.5 hour

EE

Confidential and Proprietary—DO NOT DUPLICATE

Ion WorkflowLibrary Prep

DNAAA

CC


BB

Adapter ligation3

End repair2

Fragmentation1

Physical ShearingIon* or Other Vendors

PCR based Amplicon

Approaches

PCR1

Clean-Up2

Quantification3

Enzymatic Shearingfor genomic or

amplicons

Shear DNA

Size Selection*

Purify DNA11

22

33

25

SequencingDD

CompatibleFASTQ Data

EE

Template PrepCC

Size selection4

Nick Translation and Amplification

5

Quantification6

Total Time 2 - 6 hours

Total Time ~2 hours

*Optional for amplicon sequencing

25

Total Time 2 hours

Size Selection*

Ligation and Nick Translation

Amplification

33

44

55

10-100ng Genomic DNA

ShearDNA

Fragmented DNA(Peak ~200 bp)

Blunt End Repair

End Polished Fragments(Broad size distribution)

Ion Fragment Library Kit Fragment library Workflow

Adapter Ligation& Size Selection

Nick Translate& PCR Amplify

(A-P1 enriched through amplification)

Template Preparation


Long Mate Pair Protocols for Improved de novo Assembly Mapping, Structural Variation 2-10kb inserts. Download protocol at www.iontorrent.com/community

27

A Library Solution to Meet Every Customer’s Needs

Adapter ligation2

Fragmentation1

Ion Xpress™ Barcode

Diagenode Bioruptor™Ion Xpress™ Plus Fragment Library Kit

Adapter ligation

Size selection3

Amplification*Nick Repair and Amplification*

4

Quantification5

Ion Xpress™ Barcode Adapters 1-16

Sage Science Pippin Prep™E-Gel® SizeSelect™ Gels

Now Available from Life

• Complete library preparation for 100-400bp DNA fragments in as little as 2 hours for genomic and amplicon libraries

• Enzymatic fragmentation module removes need for physical shearing and is automation compatible (Library Builder System)

• Provides excellent coverage uniformity & allows for low input amounts of DNA (100ng), with higher yields than physical shearing methods

Ion Xpress™ Plus Fragment Library Kit

Adapter ligation2

Fragmentation1

shearing methods

• Higher yields allow for generation of amplification free libraries from 100ng of input DNA

30

Adapter ligation

Size selection3

AmplificationNick Repair and Amplification

4

Quantification5

2 Hour Workflow

End Repair and 2

FragmentationEnzymatic Fragmentation

1

• Efficient multiplexing of up to 32 libraries (2 kits of 16 barcodes per kit)

• Reduced cost per sample

• Minimal adaptor sequence

Ion DNA Barcode Adaptor 1-16, 17-32 Kits

Ligate AdaptorsEnd Repair and Ligate Adaptors

AmplificationOptional Amplification

3

Pool LibrariesNormalize and Pool Libraries

4

• Minimal adaptor sequence and robust error correction for added confidence in sample identification

• Automation Compatible

2 Hour Workflow

AB Library Builder™ System - Value Proposition

The AB Library Builder™ System simplifies next-gener ation sequencing by providing a validated, semi-automated solution for library creation. The system increases throughput a nd helps reduce labor. With appropriate iPrep protocol cards the sy stem can be used for upstream DNA/RNA purification.

Prepares up to 13 libraries per run, up to 26 libra ries per day

ION TORRENT kits: late Q2’2012

Validated for use with 5500 and SOLiD ® System

Automation of most tedious and labor intensive step s

ION Shear DNA

Adaptor Ligation

Size Select

Nick Translate

Shear RNA/DNA

End Repair

Adaptor Ligation

Size Select

Nick Translate

General Library Creation WorkflowsIndicates AutomationIndicates Automation

Life Technologies Global Service and Customer suppo rt

1) Enzymatic Shearing

2) Traditional Shearing (Covaris, Bioruptor®)

Validated for use with 5500 and SOLiD ® System

Ion WorkflowIon OneTouch™ System and Template Prep Kits

11

DNAAA


BB

37

Set up Amp reaction22

Start system33

Set up system11

SequencingDD

Template PrepCC


EE

*Q4 – 3.5 hours for Ion OneTouch System runs. 30 mins for OneTouch ES

Total Processing Time ~4 hours*Hands-On Time ~20 min .

The Ion OneTouch™ System

OneTouch™ Instrument and Enrichment System

Set Up Amp Reaction

Set Up Instrument

2

1

• Easy-to-use, benchtop system that automates Ion’s proven template prep protocol

• Minutes of hands-on time, 4 hours total time

• Running costs comparable to existing Ion Xpress™ template prep costs

38

Qubit Fluorimeter Qubit Fluorimeter

Reaction Reaction

Amplify

Set Up ES

Retrieve Sample

Enrich

3

4

5

6

Revolutionary In-Line PCR Technology

Denature

Prime and Extend

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Automated Enrichment Technology

Biotinylated Template + ISP

MyOne Bead + Streptavidin

Non-Templated ISP

40

Ion WorkflowSequencing and PGM Run

Anneal Sequencing Primer20 min. (10 min. hands-on)

11

PGM Setup for 2 runs

Initialize PGM and Prepare Solutions25 min. total 10 min. hands-on

Perform PGM Cleaning15 min. total 5 min.hands-on

DNAAA

CC


BB

42

Perform Sequencing Run4 - 90min (5 min. hands-on)

44

20 min. (10 min. hands-on)

Perform Polymerase Binding10 min. (5 min. hands-on)

22

Load Ion Chip20 min. (5 min. hands-on)

33Sequencing

DD

Template PrepCC


EE

Total Processing Time 90 minsHands-On Time ~30 min.


Ion Control Kits for PGM™ Workflow

Ion Sphere Quality Control Kit• Pre- and post-enrichment quality control of Ion

Spheres• Ready-to-use reagents contain Fam™ and Cy-

5® Dye labeled primers targeting Ion adaptors• Uses Qubit® 2.0 Fluorometer

Ion Control Kit • Library Control • Template Control• Sequencing Control

E.coli DH10b Control gDNA

Ready-to-use reagents for quality control of every step

43

Template Sequence AnalysisLibraryTemplate Sequence AnalysisLibrary

E.coli DH10b Control Library

Control Ion Sphere™ Particles

Analysis Workflow

DNAAA

CC


BB

11

44

Template PrepCC

SequencingDD


EE

Data Transfer from PGM Run10 min.

11

Convert Raw Signal to Base Calls60 min.

22

View Run Quality Data and Download Base Calls5 min.

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Total Processing Time ~60min.


Data Flow

Torrent Suite

29 GB 60 MB(FASTQ)

Torrent Browser

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Ion Torrent PGM™

Sequencer

Torrent Server and Database

End UserComputer

(FASTQ)

Single run analysis Multiple run analysis

To BasesRun

AssessmentData Delivery

(SFF or FASTQ)


Torrent Server Analysis Pipeline

• Process raw “DAT” data into a reads file (eg SFF, FASTQ)

• Compute run QC metrics

• Generate summary reports

DAT ProcessingDAT Processing

ClassificationClassification

Signal ProcessingSignal Processing

• Generate summary reports

• Warehouse results

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Base CallerBase Caller

Read FilterRead Filter

Alignment QCAlignment QC

Torrent Suite Data Analysis Flow

Incorporation for 1 Flow (DAT)

Incorporation over many flows (DAT)

Processed

Raw signals per flow (WELLS)

Flow space converted to base space (FASTQ)

0.1 1.2 0.3 2.1 0.1 0.2 2.1 3.1 0.00.2 2.1 3.1 0.0 0.1 1.2 0.3 2.1 0.0 0.0 0.0 3.2 1.4 0.1 1.3 1.0 0.2 0.1

474747

Processedincorporations (SFF)Flow space converted to base space (FASTQ)

0 1 0 2 0 0 3 0 0 1 0 4 0 1 0 3 0 22 0 0 1 0 0 0 3 0 4 0 1 1 0 2 0 0 30 0 3 0 4 0 4 0 1 0 3 0 2 0 0 0 1 1

@7D8NM:4:9GGGATCAGGCTGTCGAACGCGTGATTACATCTAGCTA+AA*ABBBB?BBBBBBBABBB@@@BB?BABABCDA!@$

binary

BAM Variant Call Format (VCF)##FORMAT=<ID=DP,Number=1##FORMAT=<ID=HQ,Number=2#CHROM POS ID REF ALT QUAL FILTER 20 14370 rs6054257 G A 29 PASS

Torrent pipeline – Data sizes E. coli*

GBRaw Voltage Data DAT 147GB

GBSignal Processing WELLS 8.6GB

GBBase Calls - Flow SSF 5.0 GB

271 GB

16 GB

11GB

Process Description File Type 314 chip 316 chip 318 chip

GBBase Calls - Flow SSF 5.0 GB

GBBase Calls - Base FASTQ 1.3 GB

11GB

2.6GB

GBBase Calls - Aligned BAM 2.2 GB 4.4GB

*2.0 v run 200bp runs (520 flows, 130 cycles), Dec 2011

• Worry-free automated archiving of data• Built-in reference management

Data Management

494949

Monitor Storage Space Simple Reference ManagementQuickly Flag Runs For Archiving

http://TorrentServerURL

Torrent BrowserPlan Runs, Review Quality Reports via web interface

5151

PlanRuns

Set UpReferences

ManageSequencing Runs

& Analyses ReviewAnalysis Plugin

Results

Torrent Browser: Run / Analysis Reports

Complete Run Report

525252

Simple Workflow for Mutation Detection

Integrated and automated detection & identification of mutations

– SNPs, Indel’s plug in– Realignment’s plug in (second ref:

identify/compare new data)– Plugin can be configured to

automatically execute after every analysis

View all mutations in sortable and searchable tables

– View diagnostic plots for QC

One click link to Integrative Genome Viewer or export VCF files to any 3rd party tool

– http://www.broadinstitute.org/igv/home

Uses community standard Samtools

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Expanding the Capabilities of Torrent Suite

Torrent BrowserIon PGM™ sequencer

Torrent Server Run Reports

SFF, FASTQ, BAM

Variants

57

Desktop Analysis

Alignment (TMAP )

Torrent Circuit

(Ion Community)

RNA-seq (IsoEM)U. Connecticut

De novo assembly (MIRA)

Variant Annotation (SNPeff)Edge Biosystems

Plug-Ins Cloud Analysis

NextBioXfr(Plug-In)

Torrent Circuit & Torrent Suite PluginsYour application. Your data. Your Plugin.

Assembler IsoEM

Torrent Circuit

58 58

Assembler IsoEM

snpEff NextBioXfr

de novo Assembly RNA-Seq Analysis

Variant Annotation Interpret Results

• MIRA open-source assembler

• Generate common assembly metrics

• Supports barcoded runs

• Calculate gene and transcript level expression estimates

• Isoform prediction and visualization

• Annotates and summarizes DNA variants

• snpEff open-source tool

• Supports Torrent Variant Caller

• Direct link to your NextBio Account

• Automatic upload to cloud

• One click launch into NextBio

The App Store for Torrent Suite

Your Application. Your Analysis. Your Plugin

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Ion Performance & Data

Very uniform coverage


Semiconductor Scalability – 100X in the First Year10Mb to 1Gb in one year (100x) — The Chip is the Machine™

Ion 316™ Chip

Ion 318™ Chip*

▲ Ion 316 >850 Mb▲

▲ Ion 318 >1400 Mb

▲Specification Dec 2011 R&D performance

Ion 314™ Chip

Ion 316™ Chip

*Some products have not yet been officially released and information about those products is subject to change without noticeSept 2011 data for 314 and 316 chips based on internal R&D runs

▲ Ion 314 >150 Mb▲

Ion Community Challenge

68

Ion Community Challenge

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SpeedFastest next-gen workflow

*

70

*

Growing Throughput: Length vs Density

| | 5/4/2012

Double length Double density

Double reagentsDouble instrument runtime

Same reagentsSame instrument runtime

An Integrated Semiconductor Device Enabling Non-optical Genome SequencingRothberg J.M. et al Nature doi:10.1038/nature10242 (available at www.iontorrent.com)

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Rapid Increase in Numbers of Reads

4 000 000

5 000 000

6 000 000

7 000 000

Rea

ds

Ion 318 100Q20 Reads


75

0

1 000 000

2 000 000

3 000 000

Rea

ds Ion 318 100Q47 Reads





400

500

600

Longest Perfect Reads

Rapidly Improving Read Length

341 bp read

Nothing gets better faster

525 bp read

449 bp read

0

100

200

300

August 2011 Projected Read Length

341 bp read

265 bp read

Today

525 Base Perfect Read

CGCTAAGTAATATTCGCCCCGTTCACACGATTCCTCTGTAGTTCAGTCGGTAGAACGGCGGACTGTTAATCCGTATGTCACTGGTTCGAGTCCAGTCAGA

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


1 100

GGAGCCAAATTCTAAAAATTCGCTTTTTTAGCGCAATGTCACTGACCTTAGTTGAACATTGTTTTTTAACGGATAGCGGGTTTTTAACATCTTAAGCGCC

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


101 200

CTCGACCTTTATGGTTGAGGGCGTTTTGCTATGAACGCCATCACCATTTTCCCCTCGATTATAAAACTTGAGTTATTCAGTAGTCTCCCCTCTTGCAACT

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


201 300

77

201 300

CACACCCAAAACTGCCTAACGAAAAGTTATTAATTTTCAATCATATTGCTATCAGTATTTACATTTTTTCGCTGTGCTAGAAAGGGCGCATTTATGTTAG

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


301 400

CTCGTTCAGGGAAGGTAAGCATGGCTACGAAGAAGAGAAGTGGAGAAGAAATAAATGACCGACAAATATTATGCGGGATGGGAATTAAACTACGCCGCTT

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


401 500

AACTGCGGGTATCTGTCTGATAACT

|||||||||||||||||||||||||


501 600

Ion Torrent Internal Data

525 Base Perfect Read


||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


1 100


||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


101 200


||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


201 300

78

201 300


||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


301 400


||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


401 500


|||||||||||||||||||||||||


501 600

Cum

ulat

ive

Per

-Bas

e A

ccur

acy

98

98,5

99

99,5

100

2010Q4

Simplicity Enables Accurate ReadsImproving per-base accuracy across the last five quarters

Dan Koboldt, may 2011 V1.4

Oct 2011 V1.5

Jan 2012 V2.0


Position

Cum

ulat

ive

Per

95

95,5

96

96,5

97

97,5

0 20 40 60 80 100 120 140 160 180 200

2011Q1

2011Q2

2011Q3

2011Q4

Ion Acquisition aug 2010

PGM launch Jan 2011 V1.0

Homopolymer Performance Over Time

98,0%

98,5%

99,0%

99,5%

100,0%

Bas

e A

ccur

acy

2010Q4

Nothing Gets Better FasterJan 2012 V2.0


81

95,0%

95,5%

96,0%

96,5%

97,0%

97,5%

1 2 3 4 5

Per

-Bas

e A

ccur

acy

HP Length

2011Q1

2011Q2

2011Q3

Lowest Substitution Error Rates with Ion Semiconductor Sequencing Ion 316™ Chip 200bp runs - E.coli DH10b substitution errors

85

Read Length

103

Customer Feedback

Ion Torrent Retrospective 2011January 4th, 2012 by Justin Johnson

……The One Touch has cut down our preps

…..Our latest run, still with no paired end data and still at 100bp, generated N50s of 85K and a largest contig size of 247K with an average consensus quality of 62..

No other new machine in my 10 years of working in this field has so simply just worked –straight out of the box.

104

……The One Touch has cut down our preps times, and the introduction of the Torrent Suite of Software right from the initial launch of the platform have saved us cold, hard cash.…..”

http://www.edgebio.com/blog/?p=842#more-842

BioLektures

“The plots from the long read data set shows the massive improvements made in just a few months. This makes me very optimistic for the future….”

”

August 28 2011 by Monkol Lek

105

http://biolektures.wordpress.com/2011/08/28/ion-torrent-rapid-accuracy-improvements/

107

Ion Applications

Microbial sequencing• Accurate, fast bacteria and virus de-novo & resequencing

Mitochondrial sequencing• Highly multiplexed mitochondrial sequencing for research, clinical, and forensic applications

Amplicon sequencing• Multiplexed amplicon sequencing for rapid detection of germline and somatic mutations

Custom or fixed content amplicon panels for targeted resequencing by ultra-high multiplex PCR

Supported Applications

multiplex PCR• Revolutionary Ion AmpliSeq™ Target Selection technology simplifies targeted resequencing for

research and clinical applications

Custom targeted resequencing by target enrichment• Fast and simple workflows optimized for all major target enrichment providers

Validation of whole genome and whole exome mutation• Orthogonal technology to validate SOLiD® System/Illumina whole genome/whole exome results

Library assessment• Rapid library complexity validation/QC prior to run on high throughput sequencing platforms

RNA-Seq• Affordable, fast and simple RNA-Seq solution (Initially focused on small RNAs & low complexity

transcriptomes)

Whole-transcriptome human RNA-Seq• New RNA-Seq kits featuring faster workflow and lower RNA input for human whole transcriptome

analysis• Simplified and intuitive data analysis tools to make seamless transition from microarrays

Chip-Seq• Fast and affordable analysis of DNA binding proteins target sequences

Supported Applications

Copy number detection• Accurate targeted copy-number detection for basic and clinical research application

Resequencing and de novo sequencing of E.coli DH10b Resequencing and de novo sequencing of E.coli DH10b

Microbial Sequencing

• Highly uniform coverage (equivalent to predicted) allows more efficient sequencing 32X

(314)

110

• Up to 99.999% consensus accuracy

• Data sets available @ www.iontorrent.com/community

(314)

300X(318)

Rapid sequencing, de novo assembly & identification of novel microbial strains.Rapid sequencing, de novo assembly & identification of novel microbial strains.

E. coli Outbreak Characterized Using Ion PGM™ Sequencer in 3 Days

MondayMay 30*

Library preparation

O104:H4 and HUSC41 samples (reference) strain libraries prepared

TuesdayMay 31

Sequencing runs 0104:H4 amplified and sequenced 2 x 2 runs (Ion 314)

“The biggest advantage [of the PGM] from my point of view as a public health official is that it's speedy, and speed is what is needed at the moment,”

May 31 2 x 2 runs (Ion 314)

WednesdayJune 01

Sequencing runs 0104:H4 sequenced3 x 2 runs (Ion 314)

ThursdayJune 02

Assembly Draft Genome identified, Assembled, Submitted and Released from NCBI

FridayJune 03

Assay Design TaqMan Assays Designed

*May 22 CEDC reports significant increase in patients with hemolytic uremic syndrome

Prof. Dr. Med Dag Harmsen, University Hospital Muenster

“ [The PGM] takes the shortest time to generate genomic data.”

Junjie Qin, BGI

Assembly of K. Pneumoniae Draft Genomes

Strain 241 Strain 287

perc A 21 21

perc C 28 28

perc G 28 28

perc T 21 21

perc N 0 0

Sum contig length 5616133 5535244

Num contigs 414 581

Mean contig length 13565 9527

Median contig length 6343 3996

N50 value 28754 22772N50 value 28754 22772

Max 158952 90135

strain_1191100241 “It was essential to quickly bring together the right people and resources, so that we were able to respond to the potential spread of this multi-resistant bacterium among patients in Dutch hospitals. We are especially pleased about the role of rapid whole genome sequencing of the outbreak strain.”

Hajo Grundmann, epidemiologist at the RIVM

Recent Publications

Enterohemorrhagic E. coli Shiga-Toxin–Producing E. coli

Alexander Mellmann, Dag Harmsen,

Craig A. Cummings et al.

Mark Pallen, Junjie Qin, Ph.D et al.

Links at www.iontorrent.com/community

Rapid Economical Sequencing Enables New Types of Experiments

Day isolated Daptomycin Vancomycin Designation

32 susceptible Sensitive VSSA

52 susceptibleHetero-

intermediatehVISA

Metric MRSA C. Dip

Total reads 1,694,550 921,464

Total Mbp 230.76 105.62

Q17133.76 76.85

Longitudinal studies on isolated MRSA samples for assessment of drug resistance mutationsLongitudinal studies on isolated MRSA samples for assessment of drug resistance mutations

• 5 MRSA strains isolated from same individual across different time points

• Vancomycin resistance increases• Daptomycin resistance appears and

disappears

56Non-

susceptibleIntermediate VISA

83 susceptible Intermediate VISA

109 susceptible Intermediate VISA

Q20 101.83 63.32

Contigs(De Novo)

516 189

N50 (bps) 10051 24138

Assembly Size 2,775,167 2,487,483

GC Content ~32% ~53.5

Data Courtesy: Prof Sean Grimmond & Jason Steen

• Results from single 316 Runs

In between lines of codeBiology, sequencing, bioinformatics and moreIon Torrent Mate Pairs and a single scaffold for E coli K12 substr. MG1655 March 2, 2012

ConclusionsIon Torrent seems to have done a good job in enabling mate pair sequencing on their platform, with nice tight size distributions, and impressive throughput relative to 454. These mate pair reads, together with the shotgun (s ingle end) reads, are very useful for de novo assembly . The de novo assembly approach Ion Torrent chose, using sff_extract, MIRA and SSPACE, seems to

115

approach Ion Torrent chose, using sff_extract, MIRA and SSPACE, seems to be giving quite long contigs, with almost all genes complete. However, newbleroutperfoms SSPACE in scaffolding. Newbler is able to assemble the reads into a single scaffold, even with shotgun reads only supplemented with the 8.9 kb mate pairs. However, newbler’s algorithm is not able to produce as long contigs as MIRA does. So, a viable strategy for de novo assembly, using Ion Torrent shotgun (single end) plus mate pair reads, would be to generate contigs with MIRA, contigs and scaffolds with newbler, and elongate the newbler contigs with the MIRA contigs to reduce the number of gaps in the newbler scaffold(s).

Amplicon Sequencing

Rapid amplicon sequencing using enzymatic fragmentation approachRapid amplicon sequencing using enzymatic fragmentation approach

• 2 hour library workflow with 100ng of input DNA from Coriell cell line rs2247528 GG

9984X

Proof of principle using enzymatic fragmentation

input DNA from Coriell cell line GM04671

• Average coverage of 4,600x with 10 existing 575bp amplicons on Ion 314™ Chip

• 5 germline SNP variants identified consistent with dbSNP

rs2247528 GG

rs2277265 GG

rs1292089 AA

rs1800775 AC

rs2070971 GT

12366X

11039X

4426X

3836X

FALCON Application Development Team, Life Technologies

Ion AmpliSeq™ Technology: As Simple As PCR

+

Up to1536 primer pairs

Single-tube, ultra-high multiplex for targeted resequencing

123

Construct Library

3.5 hours

Prepare Template Run Sequence Analyze Data

10 ng DNA

Ion AmpliSeq™ Technology

How does it work?

124

2 x 100bp Paired End Sequencing Protocol & app note available at www.iontorrent.com/community

• 5X reduction in indel errors to 0.19% 10X improvement in consensus accuracy

• Unique combination of Long Reads and Paired End Sequencing

• Potential for 2 x 200bp paired end reads

127

Simple Library Construction for Paired End Sequencing

Generate Inserts

LibraryConstruction

2 hours

Construct Library

2 hours 2 X 1.5 hours4 hours 0.5 hour

Add new P1-Paired- End adapter containingspecific nicking site

[See protocol for details]

Ion Xpress Plus Fragment Library Kit

Library Insert Size 130bp or 260bp

Simple Molecular Biology for Paired End Sequencing

Forward Read On the PGM

Fill InOff the PGM

Create Reverse Template

129

Create Reverse TemplateOff the PGM

Reverse Read On the PGM

2 hours 2 x 1.5 hours

Sequence

2 x 1.5 hours4 hours 0.5 hour

Simple Data Workflow for Paired End Sequencing

Forwardfastq

Reversefastq

Pairing Plug-in

PairedSingleton Reads

(fastq)

3rd party

130

Torrent Server

fastq 3rd party analysis tools

2 hours 2 x 1.5 hours4 hours 0.5 hour

Analyze Data

0.5 hour

Ion Paired End SequencingPaired End Plug In

PairedEndPairing Plug-in

Paired ReadsSingleton Reads

(fastq)

3rd party analysis toolsPairedEndPairing.tar.gz

Fwd + Rev +Singleton

131

Single Click Plug-in creates files to be utilized by 3rd

party software

High Pairing Rate for 2 x100 Paired-End Sequencing - Ion 316 Chip E. coli Run C29-128 / C29-129 - www.iontorrent.com/community

Throughput

Fwd Rev

Total Reads 3.5M 3.2M

AQ20 Bases 440Mb 386Mb

134

AQ20 Bases 440Mb 386Mb

Perfect Bases 397Mb 349Mb

Total Perfect Bases 746Mb

Pairing Rate 92%

~AQ30 level accuracy from 2 x 100bp reads E. coli Run C29-128 / C29-129 - www.iontorrent.com/community

Accuracy

Forward Reverse

Avg. Length 119 110

Coverage @ 1X 100% 100%

135

Coverage @ 1X 100% 100%

Avg. Depth 93.9X 82.4X

Raw Accuracy (aligned) 99.5% 99.0%

Merged Raw Accuracy 99.86%

Why Perform Paired End Sequencing (PES)

Ability to sequence 2 tags from the same DNA fragment

Extension of read length,

Structural Changes, indels, de novo assembly

136

Enhanced Accuracy

Extension of read length, increases mapping in de novo assembly

**All of which enhance the number of uniquely mapped reads

De Novo Genome

Assembly

Single End Sequencing

400 bases

Paired End Sequencing

2x100 bases2x200 bases

Mate Pair Sequencing

60 base tags2-10 Kb inserts

De Novo 400 bases

2x100 bases Not used

Comparison of Sequencing Strategies

De Novo Txnome

Genome Structure

400 bases2x100 bases2x200 bases

Not used

400 bases[small features]

2x100 bases2x200 bases

[mid-size features]

60 base tags2-10 Kb inserts[large features]

Major Advantage(s) Speed

Enhance Accuracy Improve Mapping

Unique Reads/Align

Investigate large rearrangements

137

• Create whole transcriptome (WT) or small RNA libraries

• Maintains strand orientation and minimizes bias and error

• Adaptor and RT primer sequences specific for PGM sequencing allowing inputs of 200ng of total RNA or 5ng of miRNA

Ion Total RNA-Seq Kit

Adapter ligation2

Fragmentation1

138

Adapter ligation

Reverse Transcription

3

Size Selection4

Amplification5

Adapter Ligation Strategy

P

P

5’ ligationjunction

3’ ligationjunction

5’ adaptor

5’ ‘guide’

3’ adaptor

3’ ‘guide’ adaptor (3’ blocked)

RNA of Interest blocked

139Confidential and Proprietary

• 5' adaptor and 3‘ adaptor are attached in a simultaneous and directional manner.

• cDNA is produced through a separate RT primer • This strategy of directional ligation maintains strand orientation• Create either whole transcriptome (WT) or small RNA libraries

using the same kit

5’ ‘guide’adaptor

RNA of Interest blocked3’ RT primer

Ion Total RNA-Seq Kit v2: Simple & Fast Workflow

Preparation of small RNAObtain total RNA then determine the quality

Enrich small RNA

Quantitate small RNA sample and determine input amount

Preparation of whole transcriptome RNA25-500 ng rRNA-depleted total RNA or 1-500 ng poly(A)

RNA or 100 ng total RNA

Fragment the RNA

Clean up the RNA

SOLiD™ amplified library constructionHybridize and ligate the RNA adapters

Preparation of small RNAPreparation of whole transcriptome RNA

PGM ™ amplified library construction

Magnetic beads

Magnetic beads

Magnetic beads


140

Perform reverse transcription

Purify the cDNA

Assess the yield and size distribution of the amplified DNA

SOLiD™ System templated bead preparation and sequenc ing

Amplify the cDNA (BC addition)

Purify the amplified DNA

IonSphere™ particle preparation and Ion PGM™sequencin g

Magnetic beads

Magnetic beads

Magnetic beads

Magnetic beads

Magnetic beads

Magnetic beads

< 6 hours< 5 hours

Ion RNASeq: Superior Performance Versus Microarrays for Gene Expression Studies

Increased detection of human transcriptsIncreased detection of human transcripts

141Ion Torrent Internal Data

• Detection– More genes detected

• Differential Expression– More significant DEGs

Gene Expression by SequencingIon PGM™ Sequencer data exceeds microarray data at 2M reads


142

Mean mapped reads (UHRR+HBRR)

Differentially expressed genes Ion PGM

Differentially expressed genes microarrays

1,001, 951 583 4,198

2,019,947 4,630 4,198

2,890,165 4,836 4,198

3,781,665 4,944 4,198

4,813,715 4,994 4,198

Detection of Novel Exons & Alternate Splicingwith Ion PGM™ Sequencer data


143

Ion PGM™ sequencer derived RNA-Seq results from analysis of a Ewings sarcoma cell line (data courtesy T. Triche, Childrens Hospital of Los Angeles).

Detection of Fusion Transcriptswith Ion PGM™ Sequencer data

EWSR1/FLI1 fusion protein type 1 (EWSR1/FLI1 fusion ) mRNA


144

Ion PGM™ sequencer RNA-Seq results from analysis of a Ewings Sarcoma cell line (Data courtesy T. Triche, Childrens Hospital of Los Angeles)

chr22 chr11

Ion semiconductor sequencing peer reviewed publications

-An integrated semiconductor device enabling non-op tical genome sequencing.Rothberg J.M. et al. 2011, Nature. 475, 348-52.

-Open-Source Genomic Analysis of Shiga-Toxin-Produc ing E. coli O104:H4.Rohde H. et al, 2011, N. Engl. J. Med. 2011 Jul 27

-Prospective Genomic Characterization of the German EnterohemorrhagicEscherichia coli O104:H4 Outbreak by Rapid Next Gen eration Sequencing Technology.

146

Technology.Mellmann A. et al. 2011, PLoS One. 6(7):e22751.

-Genetic diversity and population structure of the endangered marsupial Sarcophilus harrisii (Tasmanian devil).Miller W. et al. 2011, Proc. Natl. Acad. Sci. U S A. 108 12348-53.

-Evolution of Multidrug Resistance during Staphyloc occus aureus Infection Involves Mutation of the Essential Two Component Re gulator WalKR.Howden B.P. et al. 2011, PLoS Pathog. 2011, 7(11): e1002359.

-Ion Torrent PGM sequencing for genomic typing of N eisseria meningitidisfor rapid determination of multiple layers of typin g information .

-Possible differentiation of cerebral glioblastoma into pleomorphic xanthoastrocytoma: an unusual case i n an infantYang M.H.M et al, 2012, Journal of Neurosurgery:Pediatrics, doi:10.3171/2012.1.PEDS11326

-Genome Sequence of the Bacterioplanktonic, Mixotro phic Vibriocampbellii Strain PEL22A, Isolated in the Abrolhos Ba nkAmaral et al,2012,Journal of Bacteriology, doi:10.1128/JB.00377-12

147

for rapid determination of multiple layers of typin g information .Vogel et al,2012,J Clin Microbiol.

-Rapid Detection of the ACMG/ACOG-Recommended 23 CFTR Disease-Causing Mutations Using Ion Torrent Semiconductor Sequ encing.J Biomol Tech. 2012 Apr;23(1):24-30, Elliott AM et al, Ambry Genetics.

-De Novo Assembly of a Filamentous Blue- Green Algal G enome Enabled by a Novel Extra-Long Read Sequencing ProtocolClancy et al,2012,LifeTechnologies

174

Ion Community

Ion Communitywww.iontorrent.com/community

• Open protocols, datasets and source code

• 6000 users growing at >150 every week

175

http://ioncommunity.iontorrent.com

>150 every week

• Prizes for contributions to the community; grants for application development

• Developer Access to pre-release products

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

© 2011 Life Technologies Corporation. All rights reserved. TaqMan is a registered trademark of Roche Molecular Systems, Inc. GeneChip is a registered trademark of Affymetrix Inc. MiSeq is a trademark of llumina, Inc. Pippin Prep is a trademark of Sage Science, Inc.

The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.

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