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Ion PGM Platform: Technology & Sequencing Applications

Mubasher “Bash” Dar, Ph.D.Sr. Applications ScientistThermo Fisher Scientific

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Ion Technology Overview of Ion Torrent™ Sequencing Technology

Applications on Ion AmpliSeq (Multiplexing PCR and RT-PCR) Sequencing Data Other Sequencing Applications

Data Analysis• Primary Analysis (Torrent Suite from Ion)

• Secondary Analysis (Commercial Vendors)

Agenda

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Personal Genome Machine® Sequencer & Chip

Easy, automatic fluid connections.Match the size of the Ion chip to your

application.

Low cost, convenient, single

use device.

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PGM™ System Orientation

Touchscreen

Chip Compartment (chip clamp/squid)

Nucleotide bottles

Waste Bottle

Wash 3 Solution

Wash 2 Solution

Wash 1 Solution

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Ion Workflow Overview

Prepare Library

Clonal AmplificationDNA / RNA

Data Analysis

Library Preparation

DNA Sequencing*

Isolate Positive Ion Sphere™ Particles

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Data Analysis

Load Chip and Sequence

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Template Preparation*

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Scalability SpeedSimplicity

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Ion Technology Core Principles

Scalability CMOS technology 40 years of Moore’s law

Simplicity Natural nucleotides No lasers No optics No camera No fluorescence No enzyme cascade

Speed Rapid detection of sequence

extension

The Chip is the Machine TM

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Scalability: Semiconductor Technology

Wafer Chip Chip Cross Section

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Simplicity: Natural Chemistry

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Speed: 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

Rothberg J.M. et al Nature doi:10.1038/nature10242

Sensor Plate

Silicon SubstrateDrain SourceBulk

dNTP

To column receiver

∆ pH

∆ Q

∆ V

Sensing Layer

H+

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Ion Torrent™ Workflow: Sequencing

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Ion Workflow – PGM™ System Sequencing Run

Perform Sequencing Run4

Anneal Sequencing Primer1

Perform Polymerase Binding2

Load Ion Chip ™ Device3

PGM™ System Setup for 2 runs (200bp or less)

Initialize PGM™ System and Prepare Solutions

Perform PGM™ System CleaningDNAA

SequencingD

Template PrepC

CompatibleLibrary Prep

B

CompatibleData

E

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Sequencing: Flows

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

Sequence of InterestKey Sequence

T A C G T A C G T

Flows 1-4 …9-12Flows 5-8 … etc.T

A

C

GFlows 1-4

...---Ion Sphere™ Particle

• 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’

C G A CG T AC GT A

A “cycle” is four consecutive dNTP flows: for instance, T-A-C-G = 1 cycle

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T

• 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’

Sequencing: Flows

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

Sequence of InterestKey Sequence

C

T A C G T A C G T

Flows 1-4 …9-12Flows 5-8 … etc.T

A

C

G

Flows 5-8

...---Ion Sphere™ Particle

C G A CG T AC GT A

A “cycle” is four consecutive dNTP flows: for instance, T-A-C-G = 1 cycle

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TIon Sphere -----Primer------ A G T C A A G C G T C C C A T G

Sequence of InterestKey Sequence

C

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

Flows 1-4 …9-12Flows 5-8 … etc.T

C

G

T

Flows 9+

AG...---Ion Sphere™ Particle

• 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

T A

A “cycle” is four consecutive dNTP flows: for instance, T-A-C-G = 1 cycle

Sequencing: Flows

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TIon Sphere -----Primer------ A G T C A A G C G T C C C A T G

Sequence of InterestKey Sequence

AC G

T A C G T A C G T

Flows 1-4 …9-12Flows 5-8 … etc.T

A

C

G

T

T T CGCA GGGT AC

And so on…

...---Ion Sphere™ Particle

• 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’

C G A CG T AC GT A

A “cycle” is four consecutive dNTP flows: for instance, T-A-C-G = 1 cycle

Sequencing: Flows

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Throughput of PGM Sequencer -- Chip type Reads

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Ion PGM™ Metrics

Ion 314™ Chip

Ion 316™ Chip

Ion 318™ Chip

Ion PGM™ Chip Ion User Q20 bases

314 cnservice.ion 167,753,95

316 Corebotz 789,113,176

318 cnservice.ion 1,355,996,615

External PerformanceWell Beyond Spec(Ion CommunityRecogntION Runs)

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Ion Torrent™ Workflow: Template Prep

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Ion Workflow – Template Prep

Enrich – Ion OneTouch™ ES

DNAA

SequencingD

Template Prep

C

CompatibleLibrary Prep

B

CompatibleData

E

Qubit ®Fluorometer

Total Processing Time ~5-8 hoursHands-On Time ~25 min.

Amplify, recover – Ion OneTouch™ 2 Instrument

3

Assemble amplification reaction

2

1

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Ideal Clonal Amplification

Amplification

10

10

10

10

10

10 1 0

10

1 010

10

Expanded view

PrimersdNTPsPolymeraseMgCl2

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PostAmplification

Add MagneticStreptavidin 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!

Ion Sphere™ Particle Enrichment

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Ion Torrent™ Workflow: Library Prep & Ion PGM Publications Data

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Ion AmpliSeq Technology – Multiplexing PCR and RT-PCR

AmpliSeq DNA

Ready-to-Go Panel (e.g. Cancer)

Custom Panels (Any Genes)150, 200 or 375bp

AmpliSeq RNA

Ready-to-Go Panel (cancer & Lung)

• 150bp panels are FFPE compatible• 10ng of DNA or RNA required

Whole Transcriptome

Whole Exome

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Ion AmpliSeq™ Library Workflow

• Prepare your samples using standard PCR techniques

• Starting with just 10 ng of input DNA – compatible with FFPE samples

• Leverage Ready-to-Use panels or build your own Custom panel

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Ion AmpliSeq™ Ready-To-Use Panels

Cancer Hotspot Panel v2

50 genes >2,800 COSMIC Mutations 207 Amplicons

Comprehensive Cancer Panel

409 Genes 16,000 Amplicons

Inherited Disease Panel

700 Diseases 328 Genes 10,000 Amplicons

www.ampliseq.com

Custom AmpliSeq Panel of your genes

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Ion PGM™ Sequencer leads the market in SNP accuracy (>90% of variation)

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PGM Sensitivity and Mutation Detection

Mutation Type

PGM Sensitivity

SNP 1-5%

INDELS 20%

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Sequencing of circulating cell free DNA suggests non-invasive profiling could be used instead of metastatic biopsies

• Circulating cell free DNA (cfDNA) profiled using the Ion AmpliSeq™ Cancer Hotspot PanelInstead of metastatic biopsies, sequencing on the Ion PGM™ System was used to screen metastatic breast cancer tumor (n=69) and serum (n=31) samples from 17 patients

• Allele frequencies as low as 0.5% were detected 13 out of 17 (76%) had concordant tumor and plasma mutation findings

• 92% of findings validatedIllumina technology was used to confirm 77 (92%) out of 84 samples tested. Two patient’s tumor mutations were detected in the plasma by Illumina sequencing that were not detected by Ion Torrent sequencing

• Rapid, cost-effective and non-invasive method could improve patient screening“This represents a non‐invasive approach, at a reasonable cost, with a quick turnaround time. Consequently, it might increase patient access to molecular screening programs and lead to personalized treatment approaches.”

http://annonc.oxfordjournals.org/content/early/2014/07/25/annonc.mdu288.abstract

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Sequencing platform comparison for solid tumor genomic biomarker discovery

• Solid tumor genomic biomarker discoveryProspective phase I clinical trail patients samples were sequenced

• Sequencing platform comparison27 samples (FFPE and fresh frozen) were compared between the MiSeq (TruSeq Amplicon Cancer Panel) and the Ion PGM™ System (Ion AmpliSeq™ Cancer Hotspot Panel) as well as Sequenom MassARRAY (OncoCarta 1.0)

• 97% concordance between MiSeq and PGM variant callsAt an allele frequency ≥13% and depth of coverage of ≥500% (found 91% concordance between MiSeq and Sequenom). 21 out of 27 samples showed a 100% concordance (78%) between MiSeq and PGM

• Similar variant allele frequencies between MiSeq and PGM “Variant allele frequencies of gene mutations detected on both NGS platforms were strikingly similar.”

• Actionable mutation discovery57% patients (53 out of 93) were allocated clinical trials due to actionable mutations

http://dx.doi.org/10.1038/bjc.2014.350

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Evaluation of a New High-Throughput Next-Generation Sequencing Method Based on a Custom AmpliSeq Library and Ion Torrent PGM Sequencing for the Rapid Detection of Genetic Variations in Long QT Syndrome• Custom AmpliSeq™ panel and the Ion PGM™

System were used for rapid variant detection in long QT syndrome

• The AmpliSeq™ panel was designed to the five most prevalent cardiomyopathy-causing genes (KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2)

• Covering 86% of the targeted regions

• Using 30 previously characterized samples, all variants (40 SNVs, 17 indels) were detected

• NextGENe v.2.3.4 (SoftGenetics) & Alamut 2.3.6 were used for further signal processing, base calling, sequence alignment, and variant analysis

• The future: • “Studies are ongoing to improve this approach by obtaining a

more complete, faster, and cheaper molecular exploration of patients presenting with LQTS. First of all, use of either an Ion 318TM (PGM sequencer) or PITM (Proton sequencer) chips would allow analysis of more patients simultaneously in the same run. Secondly, as illustrated by the recent availability of the AmpliSeqTM exome, improvement of algorithms used by the AmpliSeqTM designer would allow exploration of an increasing number of LQTS-causing genes, for which all targeted sequences will be efficiently covered.”

http://dx.doi.org/10.1007/s40291-014-0099-y

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Routine use of the Ion Torrent AmpliSeq™ Cancer Hotspot Panel for identification of clinically actionable somatic mutations

• Evaluation of AmpliSeq™ Cancer Hotspot Panel v2 for somatic mutation detection

• Samples used: cell line DNA; FFPE cell line DNA with genetically engineered mutations; FFPE samples with known mutations

• “Our results suggest that >100× coverage is needed to identify somatic mutation results with confidence.”

• Accuracy studies demonstrated 100% concordance

• Variant concordance was >95% between runs

• “The sources of disparity between variants called for the same library across the three chips were low variant frequency and homopolymeric sequences, both easily masked based on our decision tree.”

• Limit of detection was found to be 5% for SNVs and 20% for indels

• “Importantly, we were able to identify two additional actionable EGFR mutations (T790M) from this cohort that are not included in our single gene assay as it currently stands.”

• “We also showed that input DNA concentrations well below those recommended by the manufacturer could result in adequate sequencing reactions. We have tailored the runs to produce robust analyses with available starting material of as little as 1 ng of DNA isolated from FFPE tissue.

http://dx.doi.org/10.1515/cclm-2013-0883

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Hereditary hearing loss: a 96 gene targeted sequencing protocol reveals novel alleles in a series of Italian and Qatari patients

• Custom AmpliSeq™ panel targeting 96 genes implicated in hereditary hearing loss was sequenced using the Ion PGM™ System• Planned release as a Community Panel

• 12 families from Italy and Qatar were assessed with detection of a pathogenic mutation identified in 4 families (33%)

• Five novel alleles were uncovered in LOXHD1, TMPRSS3, TECTA, and MYO15A

• SNVs and indels with Q<20 were filtered

• SNVs present in dbSNP, with recessive pattern of inheritance and MAF>0.03, and dominant mode of inheritance were excluded

• Missense mutations were assessed using in silico tools PolyPhen-2 and MutationTaster

http://www.sciencedirect.com/science/article/pii/S037811191400328X

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Custom AmpliSeq™ panel demonstrates efficient large gene genotyping and copy number detection• Common autosomal dominant disorder

Dominant loss-of-function mutations in tumor suppressor neurofibromatosis type 1 (NF1) affects ~1/3000 individuals with ~50% sporadic de novo mutations

• Challenging geneNF1 is a sequencing challenge due to its large size, multiple identical pseudo genes, and lack of mutational hotspots

• Custom AmpliSeq™ panel sequenced efficiently on the Ion PGM™ System48 barcoded samples were sequenced on an Ion 316™ Chip with 96% of targeted bases covered 100x. The custom AmpliSeq™ panel targeted 100% and 98.8% coding region of SPRED1 and NF1 genes, respectively

• 100% mutation detectionUsing a validation set of 30 samples with known mutations

• Prospective testing identifies SNVs and CNVsFound NF1 alterations in 88% of samples (246/279) and SPRED1 and NF1 found in 92% (256/279).

http://dx.doi.org/10.1038/ejhg.2014.145

Dilution experiments demonstrate good correspondence between theoretical and observed mutant allele frequencies

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Ion RNA AmpliSeq™ Ready-To-Use Panels

RNA Apoptosis Panel

267 genesTaqMan® validated

RNA Cancer Panel50 genesCorresponds to genes in Ion AmpliSeq™ Cancer Hotspot v2

RNA Custom PanelTarget any gene300 genes in single tube

www.ampliseq.com

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AmpliSeq RNA panel aids expression study of 917 iPSC-related genes with improved detection of low abundance transcripts

• Roche team validates custom AmpliSeq

RNA panel 917 human genes targeted representing ~150

biological signaling cascades sequenced on the Ion

Proton™ System

• Stem cell differentiation model systemInduced pleuripotent stem cells (iPSC) differentiated to

cardiomyocytes used to assess custom AmpliSeq™

RNA panel

• Comparison to microarray and RNA-seqILMN Human Beadchip and Ion Proton™ System RNA-

Seq data generated for comparison

• AmpliSeq RNA panels fill important

research gap“Adaptation of AmpliSeq-RNA technology to high-

performance semiconductor technology closes an

important gap in custom RNA analysis because it

allows for the first time multi-parallel expression

analysis of hundreds of genes in up to one hundred

samples covering a dynamic range of five orders of

magnitude.”

AmpliSeq RNA-only DEGs

PM:24997760

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Ion PGM – other library types

• 16S ribosomal RNA profiling (bacteria typing)• ChIP-Seq• Viral (RNA and DNA) Sequencing – HIV & Flu• Mitochondrial DNA sequencing• Microbial Sequencing• Reproductive Medicine (Targeted gene sequencing)• microRNA (50% of known miRNA in mirBASE detected)

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16S rRNA profiling of meconium identifies bacteria correlated with premature birth

• 16S rRNA profiling on the Ion PGM™ System was used to characterize microbial communities present on meconiumMeconium is a substance that lines the fetal intestines resulting from ingestion in utero. Meconium stool is passed by a newborn soon after birth, before an infant has started to digest breast milk

• Bacteria correlated with premature birthMeconium from 52 infants was characterized and analysis identified: “Enterobacter, Enterococcus, Lactobacillus, Photorhabdus, and Tannerella, were negatively correlated with gestational age and have been reported to incite inflammatory responses, suggesting a causative role in premature birth.”

• Gestational age has largest impact on the mecomium microbiome With mode of delivery (natural v. c-section) also having a large effect

http://dx.doi.org/10.1371%2Fjournal.pone.0090784

Behind the Bench blog post:http://ioncommunity.lifetechnologies.com/community/behindthebench/blog/2014/05/23/microbial-community-analysis-via-16s-rrna-profiling

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Torrent Suite™ to Generate BAM Files for Analysis

WELLSInstrumentUnmapped

BAM

SFF

BAM

FASTQ

VCF

Variant Calling

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Kit Type % Q30 Reads

200bp Sequencing

64-68%

400bp Sequencing

57%

Error Rates on PGM and chemistry type

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For Research Use Only. Not for use in diagnostic procedures.

Life Technologies is a Thermo Fisher Scientific brand. © 2014 Thermo Fisher Scientific, Inc. All rights reserved. TaqMan is a registered trademark of Roche Molecular Systems,

Inc., used under permission and license. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.