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Introduction to Ion Torrent
Matt Osentoski, Ph.D. - Ion Torrent Specialist
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in serving
science
1
Semiconductor/Sanger Sequencing Scenarios
Semiconductor Sequencing
Sanger Sequencing
• Multi-gene panels
• Ideal for single gene assays
• e.g. Cancer panels
• Target gene candidates
Single-gene (few amplicons)
PI3K
p53
• Many samples, multiple amplicons
• Bidirectional Sequencing plus
Depth of coverage for increased sensitivity
100x recommended for Germline mutations
1000x coverage recommended for Somatic
mutations
• Few amplicons, few samples
• Bidirectional sequencing
• Can be used to confirm
variants from PGM
2
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Ion Torrent PGM & Proton Sequencers
• Easy, automatic fluid
connections.
• Match the size of the Ion chip to
your application.
• Low cost, convenient,
single use device.
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Simple, Natural Chemistry
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Ion Workflow Overview
Sample
Preparation
DNA
Sequencing
Data
Analysis
DNA / RNA
Prepare
Library
Clonal
Amplification
Isolate Positive Ion
Sphere™ Particles
Load Chip
& Sequence
Data
Analysis
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Rapid Direct Signal Detection
dNTP
H+
∆ pH
∆Q
Sensing Layer
Sensor Plate
∆V
Bulk
Source
Drain
Silicon Substrate
To column
receiver
Rothberg J.M. et al Nature doi:10.1038/nature10242
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
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Sequencing: Flows
• A “flow” is the event of exposing the chip to one particular dNTP
(T, A, C, or G), followed by a washing step
• The flow order repeats with pattern:
• ‘TACGTACGTCTGAGCATCGATCGATGTACAGC’
T
A
C
G
T
A
C
G
T
C
T
G
A
G
C
A
T
C
G
A
… etc.
T
Flows 1-4
Flows 5-8
…9-12
A
C
G
Ion Sphere -----Primer------ A G T C A A G C G T C C C A T G
...---Ion Sphere™
Particle
Key Sequence
Sequence of Interest
Flows 1-4
A “cycle” is four consecutive dNTP flows: for instance, T-A-C-G = 1 cycle
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Sequencing: Flows
• A “flow” is the event of exposing the chip to one particular dNTP (T,
A, C, or G), followed by a washing step
• The flow order repeats with pattern:
• ‘TACGTACGTCTGAGCATCGATCGATGTACAGC’
T
A
C
G
T
A
C
G
T
C
T
G
A
G
C
A
T
C
G
A
… etc.
T
Flows 1-4
Flows 5-8
…9-12
A
T C
C
G
Ion Sphere -----Primer------ A G T C A A G C G T C C C A T G
...---Ion Sphere™
Particle
Key Sequence
Sequence of Interest
Flows 5-8
A “cycle” is four consecutive dNTP flows: for instance, T-A-C-G = 1 cycle
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Sequencing: Flows
• A “flow” is the event of exposing the chip to one particular dNTP (T,
A, C, or G), followed by a washing step
• The flow order repeats with pattern:
• ‘TACGTACGTCTGAGCATCGATCGATGTACAGC’
T
A
C
G
T
A
C
G
T
C
T
G
A
G
C
A
T
C
G
A
… etc.
Flows 1-4
T
Flows 5-8
…9-12
C
T C AG
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
Flows 9+
A “cycle” is four consecutive dNTP flows: for instance, T-A-C-G = 1 cycle
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Sequencing: Flows
• A “flow” is the event of exposing the chip to one particular dNTP (T,
A, C, or G), followed by a washing step
• The flow order repeats with pattern:
• ‘TACGTACGTCTGAGCATCGATCGATGTACAGC’
T
A
C
G
T
A
C
G
T
C
T
G
A
G
C
A
T
C
G
A
… etc.
T
Flows 1-4
Flow 5-8
…9-12
C
T C A G T T C G C A G G GT A C
G
Ion Sphere -----Primer------ A G T C A A G C G T C C C A T G
...---Ion Sphere™
Particle
A
Key Sequence
Sequence of Interest
And so on…
A “cycle” is four consecutive dNTP flows: for instance, T-A-C-G = 1 cycle
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10
200 bp
chemistry
100 Gb
250-350 M
reads*
PII
Throughput
10 Gb
60-80 M
reads
PI
1 Gb
5-6 M reads
Ion 318™
3-4 M reads
100 Mb
Ion 316™
~0.5 M reads
10 Mb
Ion 314™
200 bp and
400 bp
chemistry
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Library Prep
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Standard gDNA Library
• Basic Library
• Ligate “unknown” DNA between Ion adaptors
• Adaptors required for template prep and sequencing
• “common” currency of fragments
• Barcodes can be added to differentiate Libraries within a run
− 10 bp sequences added to A adapter
− Server groups reads based on codes for individual analysis
• Input requirement
• Minimum 50 ng of high quality DNA recommended (more is better)
• Garbage in, garbage out…
• gDNA library applications:
• Whole genome resequencing
• Mitochondrial/Microbial DNA
• De novo
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Library Prep: Fragment Workflow for gDNA
Shear
DNA
50-100 ng
or 1 ug DNA
Blunt End
Repair
Fragmented DNA
End Polished Fragments
(for mechanical
shearing only)
Adapter Ligation,
nick translation
& Size Selection
Template
Preparation
PCR Amplify
(if needed)
Optional:
Normalization
with Ion Library
Equalizer™ Kit
Amplified, Adapter Ligated Library
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Adapter Ligated
Library
What is Targeted Sequencing?
• Targeted Sequencing isolates and focuses your sequencing
on specific genes or genomic regions of interest rather than
surveying the whole genome
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Basic Amplicon Library Construction
• Normalize PCR products by
molarity
• Ion Shear® enzymatic
fragmentation process
removes the need for
physical shearing (if needed)
• Attach adaptors (P1 and A)
• Can add Barcodes at this time.
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Fusion Approach
NOTE: No Ion Library Kit is needed for Fusion Method.
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Ion AmpliSeq™ Technology: As Simple As PCR
Ultra-high multiplex PCR for targeted resequencing
• Up to 24,000 primer pairs per
tube
• Starting from just 10ng DNA
per tube
• Including FFPE and Fine Needle
Aspirates
• From DNA to annotated
variants in as little as 10 hours
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(up to 6000 primer pairs)
Intron
Exon
Multiplex PCR Ion
AmpliSeq™ Primer Pool
Partially digest primer
sequences
Adapters
OR
Barcoded Adapters
A
P1
A-BC
P1
Ligate adapters
Nick-translate & amplify
Non-barcoded library
OR
Barcoded library
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Simple workflows with Ion AmpliSeq™ technology
Ion AmpliSeq™ technology
Amplify
Gene
Panels
Exomes
Digest
Ligate
Fusions
RNA
Expression
CNV
Community
Panels
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What is 16S?
• Subunit folds to create sites for
ribosomal proteins
• 9 variable regions flanked by
conserved regions
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NGS-based 16S rRNA profiling identified triple the number of
bacteria in brain abscesses compared with culture method
• Microbes present in bacterial brain
abscesses studied
16S rRNA profiling and the Ion PGM™
System were used
• 400-base chemistry used to target
variable regions V1 and V2
http://jcm.asm.org/content/early/2014/03/20/JCM.00346-14.abstract
• Triple the number of bacteria identified
16S rRNA profiling results were compared
to culture and Sanger sequencing-based
detection
• Key pathogens for establishing brain
abscesses identified
Bacterium include A. aphrophilus, F.
nucleatum and S. intermedius
Behind the Bench blog
post:
http://ioncommunity.lifetechnologies.com/community
/behindthebench/blog/2014/05/23/microbialcommunity-analysis-via-16s-rrna-profiling
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Ion Workflow – Library Prep
DNA/RNA
gDNA /
Long Amplicon
Libraries
Compatible Library Prep
End Repair
Template Prep
Sequencing
Compatible Data
Amplification*
(Size dependent)
(for physical
shearing)
Adapter Ligation,
Nick repair
Short Amplicon
Libraries
PCR
Clean Up
Quantification
Ion AmpliSeq™
(DNA) Libraries
Multiplex
PCR
Digest Primers,
Phosphorylate
Adapter
Ligation
Purify
Amplification*
Digest Primers
Adapter
Ligation
Purify
Hybridization /
Adapter Ligation
Reverse
transcription
Size selection
Ion AmpliSeq™
RNA Libraries
Ion RNA
Libraries
Fragmentation
Reverse
Multiplex
TranscriptPCR
ion
Prepare WT
or miRNA
Size selection
(if needed)
Qualify &
Quantify
~2-3 hrs
~3 hrs
Qualify &
Quantify
~3.5 hrs
Amplification
Qualify &
Quantify
~5 hrs
Amplification
Qualify &
Quantify
~5-6 hrs
(if needed)
*extra option: Ion Library Equalizer™ Kit
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Template Prep
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Ideal Clonal Amplification
Primers
dNTPs
Polymerase
MgCl2
Amplification
Expanded view
1
0
25
1
0
Non-Ideal Cases
Amplification
“mixed” reads
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duplicate reads
Ion Sphere™ Particle Enrichment
Post
Amplification
Add Magnetic
Streptavidin Bead
Immobilize to
Magnet and Wash
Denature ISP
with NaOH
*
*This species can be minimized through proper dilution.
Proper DNA to Ion Sphere™ Particle ratio is critical!
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Simple Workflow:
Ion OneTouch™ 2 System
Ion OneTouch™ 2
Instrument
Ion OneTouch™
ES
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Ion Workflow – PGM™ System Sequencing Run
A
B
C
D
E
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PGM™ System Setup for 2 runs (200bp or less)
Perform PGM™ System Cleaning
DNA
Initialize PGM™ System and Prepare
Solutions
Compatible
Library Prep
1
Anneal Sequencing Primer
2
Perform Polymerase Binding
3
Load Ion Chip ™ Device
4
Perform Sequencing Run
Template Prep
Sequencing
Compatible
Data
Fully-Automated to Reduce Variability
Integrates Several Workflow Steps into a Single System
Set-up
•
•
•
•
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Template Preparation and Chip Loading
library samples
reagent cartridges
plastics
chips
3 April 2015
| Life Technologies™ Proprietary and Confidential
Thermal
Cycling
Output
Fully-Automated to Reduce Variability
Integrates Several Workflow Steps into a Single System
Set-up
•
•
•
•
31
Template Preparation and Chip Loading
library samples
reagent cartridges
plastics
chips
3 April 2015
| Life Technologies™ Proprietary and Confidential
Thermal
Cycling
Output
Fully-Automated to Reduce Variability
Integrates Several Workflow Steps into a Single System
Set-up
•
•
•
•
32
Template Preparation and Chip Loading
library samples
reagent cartridges
plastics
chips
3 April 2015
| Life Technologies™ Proprietary and Confidential
Thermal
Cycling
Output
Fully-Automated to Reduce Variability
Integrates Several Workflow Steps into a Single System
Set-up
•
•
•
•
33
Template Preparation and Chip Loading
library samples
reagent cartridges
plastics
chips
3 April 2015
| Life Technologies™ Proprietary and Confidential
Thermal
Cycling
Output
Run QC and Data Analysis
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From Torrent Suite on Instrument to data comparison on Ion Reporter
35 35
For Research Use Only. Not intended for any animal or human
therapeutic or diagnostic use.
Torrent Server Analysis Pipeline
DAT Processing
Classification
Signal Processing
• Process raw .DAT files into a
sequence file
• Compute run QC metrics
Base Caller
• Generate initial summary report
Read Filter
• Warehouse results
Alignment QC
Plugins
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Example Workflow: Variant Calling
1
2
3
Auto-analysis
through Run
Planning
Define analysis
at loading
Call variants on
re-analysis
Torrent Server
Ion Torrent™
PGM™
Sequencer
Torrent Server
To Bases
Torrent Suite™ Software
37
Run
Assessment
Torrent Browser
and Plugins
Data Delivery
(BAM)
Run Analysis
Alignments
Quality Scores
Variant Calls
Ion Reporter
Run Report – Summary of Unaligned reads
New Median and Mode
metrics added
Click for details of each section
38
Run Report – Chip Loading & Key Signal
Consensus Key 1-mer
Graph
Signal strength of the first
three bases of the key
Visualization of loading density
39
Run Report – Chip Well Details
Measure loading performance and enrichment efficiency
Quantify read filter metrics: assess polyclonality and low quality
reads
40
Run Report – Read Length Details
Read Length
Histogram
Histogram of read
length for all reads in
output files
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Run Report – Reads Aligned to Reference
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Easy QC with the Coverage Analysis Plugin
Plugins can be run automatically or on demand in
Torrent Suite software
43
Simplify Data Analysis with
Integrated Variant Caller in Torrent Suite Software
Links out to IGV for easy viewing of data
underlying variant calls
44
Import Data Directly to Ion Reporter™
Plugin pushes data to secure cloud automatically
when run is finished
Start a predefined workflow
• Single sample
• Paired Tumor & Normal
• Trios – Mother, Father, & Child
45
For Research Use Only. Not intended for any animal or human
therapeutic or diagnostic use.
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Ion Reporter™ Software
Simplifying your path to results
Sequence
Import
>
Select a workflow and add Ion
Reporter to any sequencing
template in Torrent Suite™
Software
Torrent Suite securely pushes
your data into Ion Reporter™
Software and kicks off analysis
workflow
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Ion Reporter™ Software
Simplifying your path to results
Sequence
>
Import
Analyze
>
Select a workflow (if not
done in Torrent Suite),
add samples, relate, and
hit go
Identify variants across 1,
2, or 3 samples.
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Ion Reporter™ Software
Simplifying your path to results
Sequence
>
Import
Analyze
>
Explore your SNPs,
InDels, and CNVs*
Every variant is richly
annotated
•
•
•
•
•
•
•
dbSNP
COSMIC
OMIM
SIFT / Polyphen
Gene Ontology
Custom Annotations
and more….
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Ion Reporter™ Software
Simplifying your path to results
Sequence
>
Import
Analyze
>
Filter
>
Filter your variants using
experimental evidence of
functional relevance
Leverage Oncomine®* and
Ingenuity® Variant Analysis
to further filter your variants
Identify the variants that are
biologically relevant
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Ion Reporter™ Software
Simplifying your path to results
Sequence
>
Import
Analyze
>
Visualize your variants
with IGV*
Browse the raw data
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Ion Reporter™ Software
Simplifying your path to results
Sequence
>
Import
Analyze
>
Filter
>
Export
>
Report
or
Export variants to spreadsheet, VCF, or text
Select relevant variants and create
interpretive report with full audit logs /
traceability
Identify TaqMan® validation assays for
variants of interest
Access analysis metadata / outputs via API*
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Ion Reporter™ Software
For discovery or routine assays of variation Ion Reporter delivers the functionality you need
Integration w/ TS
Simple User Interface
Annotation Content
• Select Ion Reporter
workflows directly from
within Torrent Suite
• No need for command-lines
• New UI coming with IR 4.0
• Rich annotation content
integrated (dbSNP,
DrugBank, ClinVar, and
more) or import custom
annotations
Variant Detection
Aneuploidy Workflow
Filter Variants
• Quickly identify somatic
or germline SNP, InDels,
and CNVs with one assay
and one workflow
• Detect large chromosomal
abnormalities from low-pass
whole genome sequencing
(0.01X)
• Quickly filter variants to
find those that are
biologically relevant
16S Metagenomics
Broad’s IGV
Data Security
• Taxonomic classification
of your 16S samples
• Interactive taxonomy
visualization
• One click access to data
visualization (SNPs,
InDels, CNVs, etc)
• Customized karyotype
view
• Role-based logins
control access to data
• Audit logs monitor who
does what / when
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Conclusion
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Ion Community - Resource for your experiments
Discussions Forum
http://ioncommunity.lifetechnologies.com/welcome
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For Research Use Only. Not for use in diagnostic procedures.
© 2012 Life Technologies Corporation. All rights reserved
The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.
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Ion Semiconductor Sequencing
Rapid, Benchtop Sequencing for All
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