Ion Torrent Semiconductor Sequencing for Life
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Ion Torrent
Semiconductor Sequencing for Life™
1Table of Contents • Ion Technology • Ion Workflow • Ion Performance &Data • Applications 3
Ion Technology 4 Confidential and Proprietary—DO NOT DUPLICATE
Disruptive Technology
Main Frame Mini Computer Personal Computer
Sanger Sequencing Next-Gen Sequencing Ion Semiconductor Sequencing
Generations are defined by who can use them.
5The Chip is the Machine™
Scalability
Simplicity
Speed
6Simple 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
– Slow cycle time
8 Confidential and Proprietary—DO NOT DUPLICATESequencing: Flows and Cycles
• 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’
T A C G T A C G T C T G A G C A T C G A
… etc
T Cycle 1 Cycle 2 Cycle 3
A
C Ion Sphere -----Primer------ A G T C A A G C G T C C C A T G ...---Ion Sphere™
Particle
G Key Sequence Sequence of Interest
Cycle 1
Do Not DuplicateSequencing: Flows and Cycles
• 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’
T A C G T A C G T C T G A G C A T C G A
… etc
T Cycle 1 Cycle 2 Cycle 3
A
TC
C Ion Sphere -----Primer------ A G T C A A G C G T C C C A T G ...---Ion Sphere™
Particle
G Key Sequence Sequence of Interest
Cycle 2
Do Not DuplicateSequencing: Flows and Cycles
• 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’
T A C G T A C G T C T G A G C A T C G A
… etc
T Cycle 1 Cycle 2 Cycle 3
C
T CAG
T Ion Sphere -----Primer------ A G T C A A G C G T C C C A T G ...---Ion Sphere™
Particle
G Key Sequence Sequence of Interest
Cycle 3+
Do Not DuplicateFast Direct Detection
dNTP DNA Ions Sequence
– Nucleotides flow sequentially over Ion
semiconductor chip
H+ – One sensor per well per sequencing reaction
– Direct detection of natural DNA extension
∆ pH – Millions of sequencing reactions per chip
– Fast cycle time, real time detection
∆Q
Sensing Layer
Sensor Plate
∆V
Bulk Drain Source To column
Silicon Substrate receiver
13 Confidential and Proprietary—DO NOT DUPLICATEPrecise Measurement of Each Incorporation
Single Well Incorporation Trace
• Fast sequencing
A few seconds per incorporation
Raw Signal
• High signal to noise
Many data points per incorporation trace
• Enables high raw accuracy
Observations
Nuc
Wash Flow Wash
Frames (15 frames = 1 second)
14 Confidential and Proprietary—DO NOT DUPLICATEUnprecedented Scalability…
100 Gb
10 Gb
1 Gb
100 Mb
10 Mb
2011 2012 2013
The content provided herein may relate to products that have not
been officially released and is subject to change without notice.Introducing the Ion Proton™ Sequencer
The Benchtop Genome Center
21 The content provided herein may relate to products that have not
been officially released and is subject to change without notice.Ion Workflow 22 Confidential and Proprietary—DO NOT DUPLICATE
Technology Summary 23 Confidential and Proprietary—DO NOT DUPLICATE
Ion Workflow
A
DNA / RNA
B
Compatible
Library Prep Compatible with existing libraries *
C
Template Prep
4 hours Automated sample preparation
D
Sequencing
1.5 hours Fast scalable DNA sequencing
E
Compatible
FASTQ Data Storage of 1,000s of runs
0.5 hour
Confidential and Proprietary—DO NOT DUPLICATEIon Workflow
Library Prep
Enzymatic Shearing PCR based Physical Shearing
A for genomic or Amplicon Ion* or Other Vendors
DNA amplicons Approaches
1 1 PCR 1 Fragmentation
Purify DNA
B 2 Clean-Up 2 End repair
Compatible 2 Shear DNA
Library Prep
3 Quantification 3 Adapter ligation
3 Size Selection*
C
Template Prep
Total Time ~2 hours 4 Size selection
4 Ligation and Nick
Translation
D 5 Nick Translation and
Sequencing 5 Amplification
Amplification
6 Quantification
Total Time 2 hours
E
Compatible
Total Time 2 - 6 hours
FASTQ Data
25
25
*Optional for amplicon sequencingIon Fragment Library Kit
Fragment library Workflow
Shear Blunt End
DNA Repair
End Polished Fragments
10-100ng Genomic Fragmented DNA (Broad size distribution)
DNA (Peak ~200 bp)
Adapter Ligation
& Size Selection
Nick Translate
& PCR Amplify
Template
Preparation
(A-P1 enriched through amplification)
26 Confidential and Proprietary—DO NOT DUPLICATELong 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
Ion Xpress™ Plus Diagenode Bioruptor™
Fragment Library Kit
1 Fragmentation
2 Adapter ligation Ion Xpress™ Barcode
Adapters 1-16
3 Size selection Now
Available
from Life
4 Nick Repair and
Amplification*
E-Gel® SizeSelect™ Gels Sage Science Pippin Prep™
5 QuantificationIon Xpress™ Plus Fragment Library Kit
• 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)
1 Fragmentation
• Provides excellent coverage uniformity & allows for low input
2 Adapter ligation
amounts of DNA (100ng), with higher yields than physical
shearing methods
3 Size selection • Higher yields allow for generation of amplification free libraries
from 100ng of input DNA
4 Nick Repair and
Amplification
5 Quantification
2 Hour Workflow
30Ion DNA Barcode Adaptor 1-16, 17-32 Kits
• Efficient multiplexing of up
to 32 libraries (2 kits of 16
barcodes per kit)
1 Enzymatic
Fragmentation
• Reduced cost per sample
2 End Repair and
Ligate Adaptors
• Minimal adaptor sequence
and robust error correction
3 Optional for added confidence in
Amplification
sample identification
4 Normalize and
Pool Libraries
• Automation Compatible
2 Hour WorkflowAB Library Builder™ System - Value Proposition
The AB Library Builder™ System simplifies next-generation
sequencing by providing a validated, semi-automated solution for
library creation. The system increases throughput and helps reduce
labor. With appropriate iPrep protocol cards the system can be used
for upstream DNA/RNA purification.
Automation of most tedious and labor intensive steps
ION TORRENT kits: late Q2’2012
Prepares up to 13 libraries per run, up to 26 libraries per day
Validated for use with 5500 and SOLiD ® System
Life Technologies Global Service and Customer support
Indicates
General Library Creation Workflows Automation
ION Shear Adaptor Size Nick
1) Enzymatic Shearing DNA Ligation Select Translate
2) Traditional Shearing Shear
RNA/DNA
End
Repair
Adaptor
Ligation
Size
Select
Nick
Translate
(Covaris, Bioruptor®)Ion Workflow
Ion OneTouch™ System and Template Prep Kits
A
DNA
B
Compatible
Library Prep
C 1 Set up system
Template Prep
2 Set up Amp reaction
D
Sequencing
3 Start system
E Total Processing Time ~4 hours*
Compatible
Hands-On Time ~20 min.
FASTQ Data
37
*Q4 – 3.5 hours for Ion OneTouch System runs. 30 mins for OneTouch ESThe Ion OneTouch™ System
OneTouch™ Instrument and Enrichment System
1 Set Up Instrument
2 Set Up Amp
Reaction
3 Amplify
Qubit Fluorimeter Qubit Fluorimeter
4 Set Up ES
• Easy-to-use, benchtop system that automates Ion’s
5 proven template prep protocol
Retrieve Sample
• Minutes of hands-on time, 4 hours total time
6 Enrich
• Running costs comparable to existing Ion Xpress™
template prep costs
38Revolutionary In-Line PCR Technology
Denature
Prime and
Extend
39Automated Enrichment Technology
Biotinylated MyOne Bead + Non-Templated
Template + ISP Streptavidin ISP
40Ion Workflow
Sequencing and PGM Run
A PGM Setup for 2 runs
DNA
Perform PGM Cleaning
15 min. total 5 min.hands-on
Initialize PGM and Prepare Solutions
B 25 min. total 10 min. hands-on
Compatible
Library Prep
1 Anneal Sequencing Primer
20 min. (10 min. hands-on)
C
Template Prep
2 Perform Polymerase Binding
10 min. (5 min. hands-on)
D 3 Load Ion Chip
Sequencing
20 min. (5 min. hands-on)
4 Perform Sequencing Run
4 - 90min (5 min. hands-on)
E
Compatible
FASTQ Data
Total Processing Time 90 mins
Hands-On Time ~30 min.
42 Confidential and Proprietary—DO NOT DUPLICATEIon Control Kits for PGM™ Workflow
Ready-to-use reagents for quality control of every step
Ion Control Kit Ion Sphere Quality Control Kit
• Library Control • Pre- and post-enrichment quality control of Ion
• Template Control Spheres
• Sequencing Control • Ready-to-use reagents contain Fam™ and Cy-
5® Dye labeled primers targeting Ion adaptors
E.coli DH10b Control gDNA • Uses Qubit® 2.0 Fluorometer
E.coli DH10b Control Library
Control Ion Sphere™ Particles
Library Template Sequence Analysis Library Template Sequence Analysis
43Analysis Workflow
A
DNA
B
Compatible
Library Prep
C 1 Data Transfer from PGM Run
Template Prep
10 min.
2 Convert Raw Signal to Base Calls
D 60 min.
Sequencing
3 View Run Quality Data and Download Base Calls
5 min.
E
Compatible Total Processing Time ~60min.
FASTQ Data
44 Confidential and Proprietary—DO NOT DUPLICATEData Flow
Torrent Suite
Torrent Browser
29 GB 60 MB
(FASTQ)
Ion Torrent Torrent Server End User
PGM™ and Database Computer
Sequencer
Run Data Delivery
To Bases
Assessment (SFF or FASTQ)
Single run analysis Multiple run analysis
45
Confidential and Proprietary—DO NOT DUPLICATETorrent Server Analysis Pipeline
DAT Processing • Process raw “DAT” data
into a reads file (eg SFF,
Classification FASTQ)
• Compute run QC metrics
Signal Processing
• Generate summary reports
Base Caller
• Warehouse results
Read Filter
Alignment QC
46Torrent Suite Data Analysis Flow
Incorporation for Incorporation over
1 Flow (DAT) many flows (DAT) Raw signals per flow (WELLS)
0.1 1.2 0.3 2.1 0.1 0.2 2.1 3.1 0.0
0.2 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
Processed
Flow space converted to base space (FASTQ) incorporations (SFF)
@7D8NM:4:9 0 1 0 2 0 0 3 0 0 1 0 4 0 1 0 3 0 2
GGGATCAGGCTGTCGAACGCGTGATTACATCTAGCTA 2 0 0 1 0 0 0 3 0 4 0 1 1 0 2 0 0 3
+ 0 0 3 0 4 0 4 0 1 0 3 0 2 0 0 0 1 1
AA*ABBBB?BBBBBBBABBB@@@BB?BABABCDA!@$
BAM Variant Call Format (VCF)
##FORMAT=Torrent pipeline – Data sizes E. coli* Process Description File Type 314 chip 316 chip 318 chip Raw Voltage Data DAT GB 147GB 271 GB Signal Processing WELLS GB 8.6GB 16 GB Base Calls - Flow SSF GB 5.0 GB 11GB Base Calls - Base FASTQ GB 1.3 GB 2.6GB Base Calls - Aligned BAM GB 2.2 GB 4.4GB *2.0 v run 200bp runs (520 flows, 130 cycles), Dec 2011
Data Management • Worry-free automated archiving of data • Built-in reference management Quickly Flag Runs For Archiving Monitor Storage Space Simple Reference Management 49 49
http://TorrentServerURL
Torrent Browser
Plan Runs, Review Quality Reports via web interface
Plan
Runs
Set Up
References
Manage
Sequencing Runs
& Analyses
Review
Analysis Plugin
Results
51Torrent Browser: Run / Analysis Reports
Complete
Run Report
52
52Simple 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
55Expanding the Capabilities of Torrent Suite
Ion Torrent Server Torrent Browser Run Reports Variants
PGM™ sequencer
SFF, FASTQ, BAM
Plug-Ins Cloud Analysis Desktop Analysis
Alignment (TMAP)
RNA-seq (IsoEM)
U. Connecticut
Variant Annotation (SNPeff)
Edge Biosystems
Torrent Circuit
De novo assembly (MIRA) (Ion Community) NextBioXfr
(Plug-In)
57Torrent Circuit & Torrent Suite Plugins
Your application. Your data. Your Plugin.
Torrent Circuit
Assembler IsoEM
• MIRA open-source • Calculate gene and
assembler transcript level
• Generate common expression estimates
assembly metrics • Isoform prediction and
• Supports barcoded visualization
runs
The App Store for Torrent Suite
de novo Assembly RNA-Seq Analysis
snpEff NextBioXfr
• Annotates and • Direct link to your
summarizes DNA NextBio Account
variants • Automatic upload to
• snpEff open-source cloud
tool • One click launch into
• Supports Torrent NextBio
Variant Caller
Variant Annotation Interpret Results
58 58Your Application. Your Analysis. Your Plugin
Ion Performance & Data 63
Very uniform coverage Confidential and Proprietary— DO NOT DUPLICATE
Semiconductor Scalability – 100X in the
First Year
10Mb to 1Gb in one year (100x) — The Chip is the Machine™
Specification Ion 318™ Chip* Dec 2011 R&D performance
▲
▲ Ion 318 >1400 Mb
▲
▲ Ion 316 >850 Mb
Ion 316™ Chip ▲ Ion 314 >150 Mb
▲
Ion 314™ Chip
*Some products have not yet been officially released and information about those products is subject to change without notice
Sept 2011 data for 314 and 316 chips based on internal R&D runsIon Community Challenge 68
Ion Community Challenge 69
Speed
Fastest next-gen workflow
*
70Growing Throughput: Length vs Density
Double length Double density
Double reagents Same reagents
Double instrument runtime Same instrument runtime
| | 5/4/2012An Integrated Semiconductor Device Enabling Non-optical Genome Sequencing Rothberg J.M. et al Nature doi:10.1038/nature10242 (available at www.iontorrent.com) 72
Rapid Increase in Numbers of Reads
7 000 000
6 000 000
5 000 000
4 000 000 Ion 318 100Q20 Reads
Reads
Ion 318 100Q47 Reads
Ion 316 100Q20 Reads
3 000 000
Ion 316 100Q47 Reads
Ion 314 100Q20 Reads
2 000 000 Ion 314 100Q47 Reads
1 000 000
0
75Rapidly Improving Read Length
Nothing gets better faster
Longest Perfect Reads
600
525 bp read
500
449 bp read
400
341 bp read
300
265 bp read
August 2011 Projected Read Length
200
100
0
Today525 Base Perfect Read
CGCTAAGTAATATTCGCCCCGTTCACACGATTCCTCTGTAGTTCAGTCGGTAGAACGGCGGACTGTTAATCCGTATGTCACTGGTTCGAGTCCAGTCAGA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CGCTAAGTAATATTCGCCCCGTTCACACGATTCCTCTGTAGTTCAGTCGGTAGAACGGCGGACTGTTAATCCGTATGTCACTGGTTCGAGTCCAGTCAGA
1 100
GGAGCCAAATTCTAAAAATTCGCTTTTTTAGCGCAATGTCACTGACCTTAGTTGAACATTGTTTTTTAACGGATAGCGGGTTTTTAACATCTTAAGCGCC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GGAGCCAAATTCTAAAAATTCGCTTTTTTAGCGCAATGTCACTGACCTTAGTTGAACATTGTTTTTTAACGGATAGCGGGTTTTTAACATCTTAAGCGCC
101 200
CTCGACCTTTATGGTTGAGGGCGTTTTGCTATGAACGCCATCACCATTTTCCCCTCGATTATAAAACTTGAGTTATTCAGTAGTCTCCCCTCTTGCAACT
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTCGACCTTTATGGTTGAGGGCGTTTTGCTATGAACGCCATCACCATTTTCCCCTCGATTATAAAACTTGAGTTATTCAGTAGTCTCCCCTCTTGCAACT
201 300
CACACCCAAAACTGCCTAACGAAAAGTTATTAATTTTCAATCATATTGCTATCAGTATTTACATTTTTTCGCTGTGCTAGAAAGGGCGCATTTATGTTAG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CACACCCAAAACTGCCTAACGAAAAGTTATTAATTTTCAATCATATTGCTATCAGTATTTACATTTTTTCGCTGTGCTAGAAAGGGCGCATTTATGTTAG
301 400
CTCGTTCAGGGAAGGTAAGCATGGCTACGAAGAAGAGAAGTGGAGAAGAAATAAATGACCGACAAATATTATGCGGGATGGGAATTAAACTACGCCGCTT
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTCGTTCAGGGAAGGTAAGCATGGCTACGAAGAAGAGAAGTGGAGAAGAAATAAATGACCGACAAATATTATGCGGGATGGGAATTAAACTACGCCGCTT
401 500
AACTGCGGGTATCTGTCTGATAACT
|||||||||||||||||||||||||
AACTGCGGGTATCTGTCTGATAACT
501 600
Ion Torrent Internal Data
77525 Base Perfect Read
CGCTAAGTAATATTCGCCCCGTTCACACGATTCCTCTGTAGTTCAGTCGGTAGAACGGCGGACTGTTAATCCGTATGTCACTGGTTCGAGTCCAGTCAGA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CGCTAAGTAATATTCGCCCCGTTCACACGATTCCTCTGTAGTTCAGTCGGTAGAACGGCGGACTGTTAATCCGTATGTCACTGGTTCGAGTCCAGTCAGA
1 100
GGAGCCAAATTCTAAAAATTCGCTTTTTTAGCGCAATGTCACTGACCTTAGTTGAACATTGTTTTTTAACGGATAGCGGGTTTTTAACATCTTAAGCGCC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GGAGCCAAATTCTAAAAATTCGCTTTTTTAGCGCAATGTCACTGACCTTAGTTGAACATTGTTTTTTAACGGATAGCGGGTTTTTAACATCTTAAGCGCC
101 200
CTCGACCTTTATGGTTGAGGGCGTTTTGCTATGAACGCCATCACCATTTTCCCCTCGATTATAAAACTTGAGTTATTCAGTAGTCTCCCCTCTTGCAACT
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTCGACCTTTATGGTTGAGGGCGTTTTGCTATGAACGCCATCACCATTTTCCCCTCGATTATAAAACTTGAGTTATTCAGTAGTCTCCCCTCTTGCAACT
201 300
CACACCCAAAACTGCCTAACGAAAAGTTATTAATTTTCAATCATATTGCTATCAGTATTTACATTTTTTCGCTGTGCTAGAAAGGGCGCATTTATGTTAG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CACACCCAAAACTGCCTAACGAAAAGTTATTAATTTTCAATCATATTGCTATCAGTATTTACATTTTTTCGCTGTGCTAGAAAGGGCGCATTTATGTTAG
301 400
CTCGTTCAGGGAAGGTAAGCATGGCTACGAAGAAGAGAAGTGGAGAAGAAATAAATGACCGACAAATATTATGCGGGATGGGAATTAAACTACGCCGCTT
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTCGTTCAGGGAAGGTAAGCATGGCTACGAAGAAGAGAAGTGGAGAAGAAATAAATGACCGACAAATATTATGCGGGATGGGAATTAAACTACGCCGCTT
401 500
AACTGCGGGTATCTGTCTGATAACT
|||||||||||||||||||||||||
AACTGCGGGTATCTGTCTGATAACT
501 600
78Simplicity Enables Accurate Reads
Jan 2012 V2.0
Improving per-base accuracy across the last five quarters
100 Oct 2011 V1.5
99,5
Per-Base Accuracy
99
Dan Koboldt, may
98,5
2011 V1.4
98
2010Q4
Cumulative Per
2011Q1
97,5 PGM launch Jan 2011Q2
2011 V1.0 2011Q3
97
2011Q4
96,5
Ion Acquisition
96
aug 2010
95,5
95
0 20 40 60 80 100 120 140 160 180 200
Confidential
and Position
Proprietary—
DO NOT
DUPLICATEHomopolymer Performance Over Time
Nothing Gets Better Faster
Jan 2012 V2.0
100,0%
99,5%
99,0%
Base Accuracy
98,5%
98,0% 2010Q4
97,5% 2011Q1
Per-Base
97,0% 2011Q2
96,5% 2011Q3
96,0%
95,5%
95,0%
1 2 3 4 5
Confidential HP Length
and
81
Proprietary—
DO NOT
DUPLICATELowest Substitution Error Rates with Ion
Semiconductor Sequencing
Ion 316™ Chip 200bp runs - E.coli DH10b substitution errors
Read Length
85Customer Feedback 103
Ion Torrent Retrospective 2011
January 4th, 2012 by Justin Johnson
…..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.
……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
104BioLektures
August 28 2011 by Monkol Lek
“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….”
”
http://biolektures.wordpress.com/2011/08/28/ion-torrent-rapid-accuracy-improvements/
105Ion Applications 107
Supported 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
• 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)Supported Applications
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
Copy number detection
• Accurate targeted copy-number detection for basic and clinical research applicationMicrobial Sequencing
Resequencing and de novo sequencing of E.coli DH10b
• Highly uniform coverage (equivalent to
predicted) allows more efficient
sequencing 32X
(314)
• Up to 99.999% consensus accuracy
• Data sets available @
www.iontorrent.com/community
300X
(318)
110E. coli Outbreak Characterized Using Ion
PGM™ Sequencer in 3 Days
Rapid sequencing, de novo assembly & identification of novel microbial strains.
Monday Library O104:H4 and HUSC41 samples “The biggest advantage [of the PGM] from
May 30* preparation (reference) strain libraries prepared my point of view as a public health official
is that it's speedy, and speed is what is
Tuesday Sequencing runs 0104:H4 amplified and sequenced needed at the moment,”
May 31 2 x 2 runs (Ion 314)
Prof. Dr. Med Dag Harmsen,
University Hospital Muenster
Wednesday Sequencing runs 0104:H4 sequenced
June 01 3 x 2 runs (Ion 314)
Thursday Assembly Draft Genome identified, Assembled,
June 02 Submitted and Released from NCBI
“[The PGM] takes the shortest time to
Friday Assay Design TaqMan Assays Designed generate genomic data.”
June 03
Junjie Qin, BGI
*May 22 CEDC reports significant increase in patients with hemolytic uremic syndromeAssembly 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 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 RIVMRecent Publications
Enterohemorrhagic E. coli Shiga-Toxin–Producing E. coli
Alexander Mellmann, Mark Pallen,
Dag Harmsen, Junjie Qin, Ph.D et al.
Craig A. Cummings et al.
Links at www.iontorrent.com/communityRapid Economical Sequencing Enables
New Types of Experiments
Longitudinal studies on isolated MRSA samples for assessment of drug resistance
mutations
Day isolated Daptomycin Vancomycin Designation Metric MRSA C. Dip
Total reads 1,694,550 921,464
32 susceptible Sensitive VSSA Total Mbp 230.76 105.62
Hetero- 133.76 76.85
52 susceptible hVISA Q17
intermediate
Non- 101.83 63.32
56 Intermediate VISA Q20
susceptible
Contigs
83 susceptible Intermediate VISA 516 189
(De Novo)
109 susceptible Intermediate VISA N50 (bps) 10051 24138
Assembly Size 2,775,167 2,487,483
• 5 MRSA strains isolated from same
individual across different time points GC Content ~32% ~53.5
• Vancomycin resistance increases
• Daptomycin resistance appears and • Results from single 316 Runs
disappears
Data Courtesy: Prof Sean Grimmond & Jason SteenIn between lines of code
Biology, sequencing, bioinformatics and more
Ion Torrent Mate Pairs and a single scaffold for E coli K12
substr. MG1655
March 2, 2012
Conclusions
Ion 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 (single
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
be giving quite long contigs, with almost all genes complete. However, newbler
outperfoms 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).
115Amplicon Sequencing
Proof of principle using enzymatic fragmentation
Rapid amplicon sequencing using enzymatic fragmentation approach
• 2 hour library workflow with 100ng of 9984X
input DNA from Coriell cell line rs2247528 GG
GM04671 12366X rs2277265 GG
11039X rs1292089 AA
• Average coverage of 4,600x with 10
4426X rs1800775 AC
existing 575bp amplicons on Ion 314™
Chip 3836X rs2070971 GT
• 5 germline SNP variants identified
consistent with dbSNP
FALCON Application Development Team, Life TechnologiesIon AmpliSeq™ Technology: As Simple As
PCR
Single-tube, ultra-high multiplex for targeted resequencing
Up to1536 primer
pairs +
10 ng DNA
Construct Library Prepare Template Run Sequence Analyze Data
3.5 hours
123Ion 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
127Simple Library Construction for Paired End
Sequencing
Generate Library
Inserts Construction
Add new P1-Paired- End adapter containing
Ion Xpress Plus Library Insert Size 130bp or 260bp specific nicking site
Fragment Library Kit [See protocol for details]
Construct Library
2 hours 4 hours 2 X 1.5 hours 0.5 hourSimple Molecular Biology for Paired End
Sequencing
Forward Read
On the PGM
Fill In
Off the PGM
Create Reverse Template
Off the PGM
Reverse Read
On the PGM
Sequence
2 hours 4 hours 2 x 1.5 hours 0.5 hour
129Simple Data Workflow for Paired End
Sequencing
Forward
fastq
Pairing Paired
Plug-in Singleton Reads
(fastq)
Reverse
fastq 3rd party
Torrent analysis tools
Server
Analyze Data
2 hours 4 hours 2 x 1.5 hours 0.5 hour
130Ion Paired End Sequencing
Paired End Plug In
PairedEnd Paired Reads
Pairing Singleton Reads
Plug-in (fastq)
3rd party
PairedEndPairing.tar.gz analysis tools
Fwd + Rev +Singleton
Single Click Plug-in creates files to be utilized by 3rd
party software
131High 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
Perfect Bases 397Mb 349Mb
Total Perfect Bases 746Mb
Pairing Rate 92%
134~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%
Avg. Depth 93.9X 82.4X
Raw Accuracy (aligned) 99.5% 99.0%
Merged Raw Accuracy 99.86%
135Why Perform Paired End Sequencing (PES)
Ability to sequence 2 tags from the same DNA fragment
Structural Changes, indels,
de novo assembly
Extension of read length,
increases mapping in de
novo assembly
Enhanced Accuracy
**All of which enhance the number of uniquely mapped reads
136Comparison of Sequencing Strategies
Single End Paired End Mate Pair
Sequencing Sequencing Sequencing
De Novo
2x100 bases 60 base tags
Genome 400 bases
2x200 bases 2-10 Kb inserts
Assembly
De Novo 2x100 bases
400 bases Not used
Txnome 2x200 bases
2x100 bases 60 base tags
Genome 400 bases
2x200 bases 2-10 Kb inserts
Structure [small features]
[mid-size features] [large features]
Major Enhance Accuracy
Investigate large
Advantage(s) Speed Improve Mapping
rearrangements
Unique Reads/Align
137Ion Total RNA-Seq Kit
• Create whole transcriptome (WT) or small RNA libraries
• Maintains strand orientation and minimizes bias and
error
1 Fragmentation • Adaptor and RT primer sequences specific for PGM
sequencing allowing inputs of 200ng of total RNA or 5ng
2
of miRNA
Adapter ligation
3 Reverse
Transcription
4 Size Selection
5 Amplification
138Adapter Ligation Strategy
5’ ligation
5’ adaptor junction
3’ ligation 3’ adaptor
junction
P
P
3’ ‘guide’ adaptor
(3’ blocked)
5’ ‘guide’ RNA of Interest blocked
adaptor 3’ RT primer
• 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
Confidential
139
and
ProprietaryIon Total RNA-Seq Kit v2: Simple & Fast Workflow
Preparation of whole transcriptome RNA Preparation of small RNA
25-500 ng rRNA-depleted total RNA or 1-500 ng poly(A)
Obtain total RNA then determine the quality
RNA or 100 ng total RNA
Magnetic
Fragment the RNA Enrich small RNA beads
Magnetic Clean up the RNA Quantitate small RNA sample and determine input amount
beads
SOLiD™
PGM ™ amplified
amplifiedlibrary
libraryconstruction
construction
Hybridize and ligate the RNA adapters
Perform reverse transcription
< 5 hours < 6 hours
Magnetic
Magnetic
beads Purify the cDNA beads
Amplify the cDNA
(BC addition)
Magnetic Magnetic
beads Purify the amplified DNA beads
Assess the yield and size distribution of the amplified DNA
SOLiD™particle
IonSphere™ System templated beadand
preparation preparation and sequencing
Ion PGM™sequencing
Confidential
and
Proprietary—
140
DO NOT
DUPLICATEIon RNASeq: Superior Performance Versus
Microarrays for Gene Expression Studies
Increased detection of human transcripts
Ion Torrent Internal Data
141Gene Expression by Sequencing
Ion PGM™ Sequencer data exceeds microarray data at 2M reads
• Detection
– More genes detected
• Differential Expression
– More significant DEGs
Mean mapped reads Differentially expressed Differentially expressed
(UHRR+HBRR) genes Ion PGM 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
Confidential
and
Proprietary—
142
DO NOT
DUPLICATEDetection of Novel Exons & Alternate Splicing
with Ion PGM™ Sequencer data
Ion PGM™ sequencer derived RNA-Seq results from analysis of a Ewings
sarcoma cell line (data courtesy T. Triche, Childrens Hospital of Los Angeles).
Confidential
and
Proprietary—
143
DO NOT
DUPLICATEDetection of Fusion Transcripts
with Ion PGM™ Sequencer data
EWSR1/FLI1 fusion protein type 1 (EWSR1/FLI1 fusion) mRNA
chr22 chr11
Ion PGM™ sequencer RNA-Seq results from analysis of a Ewings Sarcoma
cell line (Data courtesy T. Triche, Childrens Hospital of Los Angeles)
Confidential
and
Proprietary—
144
DO NOT
DUPLICATE145
Ion semiconductor sequencing peer reviewed publications -An integrated semiconductor device enabling non-optical genome sequencing. Rothberg J.M. et al. 2011, Nature. 475, 348-52. -Open-Source Genomic Analysis of Shiga-Toxin-Producing E. coli O104:H4. Rohde H. et al, 2011, N. Engl. J. Med. 2011 Jul 27 -Prospective Genomic Characterization of the German Enterohemorrhagic Escherichia coli O104:H4 Outbreak by Rapid Next Generation Sequencing 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 Staphylococcus aureus Infection Involves Mutation of the Essential Two Component Regulator WalKR. Howden B.P. et al. 2011, PLoS Pathog. 2011, 7(11): e1002359. 146
-Possible differentiation of cerebral glioblastoma into pleomorphic xanthoastrocytoma: an unusual case in an infant Yang M.H.M et al, 2012, Journal of Neurosurgery:Pediatrics, doi:10.3171/2012.1.PEDS11326 -Genome Sequence of the Bacterioplanktonic, Mixotrophic Vibrio campbellii Strain PEL22A, Isolated in the Abrolhos Bank Amaral et al,2012,Journal of Bacteriology, doi:10.1128/JB.00377-12 -Ion Torrent PGM sequencing for genomic typing of Neisseria meningitidis for rapid determination of multiple layers of typing information. Vogel et al,2012,J Clin Microbiol. -Rapid Detection of the ACMG/ACOG-Recommended 23 CFTR Disease- Causing Mutations Using Ion Torrent Semiconductor Sequencing. J Biomol Tech. 2012 Apr;23(1):24-30, Elliott AM et al, Ambry Genetics. -De Novo Assembly of a Filamentous Blue- Green Algal Genome Enabled by a Novel Extra-Long Read Sequencing Protocol Clancy et al,2012,LifeTechnologies 147
Ion Community 174
Ion Community
www.iontorrent.com/community
• Open protocols, datasets
and source code
• 6000 users growing at
>150 every week
• Prizes for contributions to
the community; grants for
application development
• Developer Access to pre-
release products
http://ioncommunity.iontorrent.com
175For 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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