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Everything You Ever Wanted to Know About Next Generation Sequencing, but were Afraid to Ask. (Well…Almost Everything)

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What is Next Generation Sequencing (NGS)? • Catch-all term that describes several distinct modern

sequencing technologies • The actual chemistry varies, but sequence data is collected

as it is being generated • Generating DNA sequence cheaper and easier • Whole genome analysis for risk/disease detection made real

xkcd.com - THIS WORK IS LICENSED UNDER A CREATIVE COMMONS ATTRIBUTION-NON COMMERCIAL 2.5 LICENSE.

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What can NGS do that we can’t do now?

• It’s all about capacity. • Human Genome Project =

1990-2003 and $3 billion • Brute force

• Now – A few days and $1000 per genome…really?

Image courtesy of genome.gov and the National Human Genome Research Institute

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How much information?

If we define: • BP = A single

sequenced base pair • Run = a single chip,

flow cell, or 96-well plate

Platform Max BP per Run CE 96,000 Ion 314™ Chip 550,000 Ion 316™ Chip 3,000,000 Ion 318™ Chip 5,500,000 Proton™ I Chip 80,000,000 Biggest one on the market

3,000,000,000

“Massively Parallel” means lots of sequencing reads happening all at the same time

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Sequences lots of short DNA pieces at the same time.

• Traditional Sequencing theoretical limit is 1000 BP

• Ion Platforms 200-400 BP • Varies for other platforms,

may be as short as 50 BP • Not a problem for HID

CE

NGS

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Great! NGS does lots of sequencing. How will all this help us catch bad guys or identify missing persons?

• Obvious use:

• Mitochondrial sequencing • More information

• What else can it do?

File reproduced in accordance with creative commons public domain license: original work by jhc, who grants anyone the right to use this work for any purpose, without any conditions, unless such conditions are required by law, derivative work by Shanel is shown. http://commons.wikimedia.org/wiki/File:Mitochondrial_DNA_en.svg

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Single Nucleotide Polymorphisms (SNP) What’s in a SNP? More information • Identity – SNP amplicons are small, good for degraded DNA • Ancestry – No match in the database? At least you can let

law enforcement know they are looking for a male of ________ descent

• Phenotype – with _______ colored eyes and ________ hair.

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Short Tandem Repeats (STR)

• A 15 is not always just a 15… • vWA, for example • Individual A 15 repeats: TCTA [TCTG]4 [TCTA]10

• Individual B 15 repeats: TCTA [TCTG]3 [TCTA]11

• Mixture deconvolution

• More information!

STRs for Forensic or Paternity use only

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Way More Information…

• How to deal with all this information? • Bioinformatics - The field of science concerning the application of

computer science and information technology to biology; using computers to handle biological information...1

• Don’t we do that already?

• Example: Trimming

• AKA getting rid of the unusable signal at the beginning and end • Ion 314™ Chip – 550,00 BP • Average read length 200 BP • 2,750 sequence reads = too many to hand trim!

1. bioinformatics. Dictionary.com. The Free On-line Dictionary of Computing. Denis Howe. http://dictionary.reference.com/browse/bioinformatics

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Ion PGM™ for HID – How does it work? Hint: It’s not magic.

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PGM™ System for HID – How does it work?

It’s a Process… 1. Library Preparation 2. Quantify 3. Ion OneTouch™ 2 System* 4. Ion OneTouch™ ES System* 5. Sample on the Chip* 6. Chip on the Ion PGM™ System 7. Data! *The Ion Chef™ System can do Steps 3-5

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Library Preparation

1. Amplify the regions of interest

2. Carve back the primers

3. Add barcodes and adapters

All this in a single tube!

4. Clean up the reaction

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Library Prep FAQs • Ion AmpliSeq™ Panel PCR - How does it compare to what

we do now? • 100+ primer sets instead of 20+ • No fluorophores = very stable primers

• Why trim the primers? • Don’t want to waste sequencing real estate on primers • Need a blunt end for the next step (ligating the adapters)

• What do the adapters do? • Barcodes let you load multiple samples onto one chip • P1 and A/X adapters will be used as primer binding sites in the next step

(OneTouch 2 emulsion PCR) • Is the clean-up step necessary?

• Yes, you need to remove all the primer parts, dead enzymes, and salts before you move on to the next step.

• Ampure® XP Beads can be automated.

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Quantify Critical Quality Control Step! • Ion Library Quantitation Kit

• Real-time QPCR • 7500 friendly

• Adding the right amount of Library to Ion Sphere™ Particles is critical

• Can use Qubit® or Bioanalyzer®, but real-time is the most accurate

• Ion Library Equalizer™ Kit not recommended at this time

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What happens to the Library next?

3. OneTouch™ 2 4. OneTouch ™ ES 5. Sample on Chip

Clonal Amplification

Load Chip Incorporate Nucleotide

Detect and Call

6. Chip on the PGM

Ion Chef™

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Ion OneTouch™ 2 Add Library and Ion Sphere™ Particles (ISP) PCR Part 2 – Emulsion PCR

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Ideal Clonal Amplification using Emulsion PCR

Isolate templated ISPs

ISP

Primer dNTPs

Polymerase MgCl2

Final Templated ISPs ready for sequencing

Emulsion droplet

P1 Barcode + A = X

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ISP

Non-Ideal Outcomes

Amplification Amplification

ISP

Polyclonal “mixed” reads duplicate reads

ISP

ISP

ISP

ISP ISP

no template no ISP

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Non-Ideal Outcomes

Quantification of the library is important!

Too much library = lots of polyclonal “mixed” reads

Not enough library = lots of empty ISPs

It’s all about balance

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OneTouch™ 2 FAQs

• Does the PCR happen to the whole sample at once? • Yes, the whole sample is injected into the amp plate and the thermal

cycling happens to all of it at the same time.

• If it’s just PCR, what takes so long? • The rate-limiting step is the collection of the ISPs from the emulsion. The

emulsion is dripped onto the bridge a little at a time, the centrifuge spins to collect the ISPs at the bottom of the collection tubes, the excess oil and buffer flow out of the top of the collection tubes, and the process is repeated until all the ISPs have been collected.

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Ion OneTouch™ ES

• Liquid-handling robot • Enriches for ISPs with

template on them

8-well strip tube (flanked by magnets)

Pipet Tip

1) ISP sample 5) Wash Solution 2) MyOne™ Beads 6) Empty 3) Wash Solution 7) Melt-Off Solution 4) Wash Solution 8) Empty

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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!

Ion Sphere™ Particle (ISP) Enrichment

Biotin

ISP

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To the Chip!

Chips Chip Cross Section

One ISP per well…mostly

Pipet sample in through this port

Chip differences -look for the “football”

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The Chip up Close

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

Sensor Plate

Silicon Substrate Drain Source Bulk

dNTP

To column receiver

∆ pH

∆ Q

∆ V

Sensing Layer

H+

Millions of Tiny pH Meters

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Chip FAQs

• How many samples can I load per chip with the HID panels? • Ion 314™ chip – up to 8 individuals • Ion 316™ chip – up to 38 individuals • Ion 318™ chip – up to 77 individuals • For the HID-Ion AmpliSeq™ Identity Panel

• Explain “the chip is the machine” • As Ion chip technology improves, you just buy new versions of the chip • You can scale your throughput level by buying different chips • Like getting a new CE array, laser, and CCD for every run

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Chip FAQs • Why am I not supposed to wear gloves when handling the chip?

• The chip is a sensitive piece of electronics. You want to electrically “ground” your body to the instrument and then handle the chip to avoid a charge difference between the instrument and the chip.

• Gloves act as an insulator. They could preserve a charge difference between the instrument and the chip, which could be discharged as a static electricity when you place the chip. This could fry the chip.

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The Ion PGM™ System • The Ion PGM™ System

• Flows a single type of nucleotide at a time over the chip

• Detects the voltage changes from the wells in the chip

• Washes the chip • Repeats a defined number of

times • Transfers data from each flow

to the Torrent Server

If the chip is the machine, then what is this thing for?

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Simple, Natural Chemistry PGM™ observes the addition of each nucleotide in real time H+ is the “ion” in Ion Torrent™

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Ion PGM™System FAQs • Is 18 MΩ water really that important?

• Yes, since the system relies on minute pH changes to detect nucleotide incorporation, the pH of the water and all solutions is critical.

• Why does the PGM™ System need Argon or Nitrogen gas? • These inert gasses exclude CO2 from the system • CO2 dissolved in water makes carbonic acid, so back to the whole pH

thing…

• What about homopolymers (lots of the same nucleotide in a row)? • Every template molecule on the ISP is wanting many of the same

nucleotide (dNTP) at once. There just aren’t enough to go around so some template molecules “get behind”.

• 8 Thymines does not equal 8x the signal of 1 Thymine. Ion has improved its data analysis software to help compensate for this.

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Data - Run Report

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Torrent Server and Torrent Suite™ Software Web-based data delivery with integrated alignment, variant calling, and data analysis plugins

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AC C170

T CCAANT C CC180

NC T AT CA T T T190

T T

T

C

Data – How does it compare to CE?

• In CE, each peak is an aggregate of lots of molecules of the same length with the same fluorophore

• Peak heights provide some idea of the relative quantities of these molecules

173-length C 176-length T and C

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Data – How does it compare to CE? • Ion Torrent™ technology counts all of the times that an

outcome was observed, so data is more like this:

Chrom Position* Genotype Coverage A Reads

C Reads

G Reads

T Reads Deletions Freq

chr1 173 CC 3485 3 3480 0 2 0 99.85 chr1 176 CT 4776 2 2280 1 2484 9 52.01

*This would be the actual mapped position on the chromosome, so it would typically be a much larger number. These have been changed to make comparison with the previous example easier.

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Plugin Output

Easy to see heterozygous vs. homozygous

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Data FAQs • What file type should I archive? • How many runs can I store?

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Incorporation for 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

Signal Processing

Base Calling

unmapped

BAM

FASTQ 4.4 GB

242 GB 12 GB

10 GB 2.5 GB

4.3 GB ~ KB

VCF Variant Caller

BAM

Alignment

SFF

Torrent Suite™ Software Data Analysis Flow

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28 GB Raw Voltage Data DAT 129 GB 242 GB

1 GB Signal Processing WELLS 7-9 GB 12-15 GB

1 GB/ Base Calls - Flow SFF 4.5-6.0 GB/ 8-10 GB/

0.2 GB Base Calls - Base FASTQ 1-1.25 GB 1.8-2.25 GB

0.1 GB Base Calls - Aligned BAM 1.7-2.0 GB 2.7-3.0 GB

*v3.0 250 bp run (500 flows) Sept2012. The information presented here is an approximation. User experiences may vary.

Process Description File Types Ion 314™ chip Ion 316™ chip Ion 318™ chip

Torrent Pipeline – Approximate Data Sizes*

2 TB

80-100 GB

100-120GB

20-30 GB

40-60 GB

Ion PI™ chip

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Process Equipment

File sizes for a typical run of 520 flows that enables 200 base pair read length. The Torrent Server has 12 TB of space with n=2 redundancy.

The information presented here is an approximation. User experiences may vary.

Data Acquisition 40 runs PGM™

Sequencer 8 runs

Raw Data 300 runs Torrent Server 65 runs

Archived Data 4100 runs Torrent Server 880 runs

Ion 314™ chip

Ion 316™ chip

Ion 318™ chip

5 runs

40 runs

530 runs

FAQ - How many runs can be stored?

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How can I make this easier/faster/better?

• Automated Sample Preparation • Automated Template Preparation • Enzyme Upgrade

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• For Research, Forensic or Paternity Use Only. When used for purposes other than Human Identification the instruments and modules of the cited software are for Research Use Only. Not for use in diagnostic procedures.

• © 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. AMPure is a registered trademark of Beckman Coulter, Inc. Bioanalyzer is a registered trademark of Agilent Technologies, Inc.

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Thank You!

Questions?

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Please note: HID-Ion AmpliSeq™ panels have not been tested on these platforms

Automated Sample Preparation

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Ion AmpliSeq™ Automation on NGS Express: $ 63.5K

http://www.perkinelmer.com/pdfs/downloads/APP_Ion_AmpliSeq_Life_Technologies.pdf

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Ion AmpliSeq™Automation on Freedom EVO® 150

• Throughput: 96 samples in 4.5hours (HSM panel) • Ion AmpliSeq™ Library Kit 2.0 384LV processes 288 samples • Demonstrated protocol available in Ion Community • $125k + $25K PCR thermocycler = ~$150K http://ioncommunity.lifetechnologies.com/docs/DOC-6536 (documentation) http://ioncommunity.lifetechnologies.com/docs/DOC-6652 (poster)

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Ion AmpliSeq™ Automation on Hamilton Starlet System

• Low or high throughput • 96 well multiprobe

heads • Full walk away

solutions to free up time for important research

• Upgradable with lab focus changes or new sequencing technologies

http://www.hamiltonrobotics.com/fileadmin/user_upload/prodb/app_notes/Genomics/BR012_NGS_US.pdf

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Automated Template Preparation

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Ion OneTouch™ 2 System for Template Prep

• Available now for both the Ion PGM™ and Ion Proton™ Systems

• Supports 200 bp and 400 bp on the Ion PGM™ System & Up to 200 bp on Ion Proton™ System

• Simple and Robust

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Ion Chef™ System Bringing Workflow Simplicity to Ion Proton™ Sequencing

• Simple to use • Automated template prep AND chip

loading – library in, loaded chips out • Simple reagent and consumables

loading– minutes of hands-on time • Minimizes potential sources of user

error and sequencing variability

• High throughput • Processes 2 chips and multiple

barcoded samples within hours

• Flexible • Supports all Ion systems, chips, read

lengths, and “Classic” and Avalanche template prep chemistry

Insert Link to lifetech catalog page https://www.youtube.com/watch?v=qpsd93d2mcc

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Ion Chef™ System Benchtop Sequencing Prep with Integrated Workflows

Tip Rack

Chip Loader

Reco

very

M

odul

es

Thermal- cycler

Reag

ent B

ay

Enric

her

Waste

Robo

tic A

rm

Ion Chef™ System Quotes Early Q2, Shipments Commence End Q2, Ship in Volume Q3

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• For Research, Forensic or Paternity Use Only. When used for purposes other than Human Identification the instruments and modules of the cited software are for Research Use Only. Not for use in diagnostic procedures.

• © 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. AMPure is a registered trademark of Beckman Coulter, Inc. Bioanalyzer is a registered trademark of Agilent Technologies, Inc.