CFTR_Ion

Report
Ion Torrent
Semiconductor Sequencing for Life™
Ion Torrent for amplicon based sequencing
1
Confidential and Proprietary—DO NOT DUPLICATE
Bench Top Convenience
Low cost, convenient,
single use device.
2
Easy, automatic fluid connections.
Match the size of the Ion chip to your application.
The Chip is the Machine™
Scalability
Simplicity
Speed
Life Technologies’ Portfolio of Complementary
Sequencing Solutions
5500 SOLiD
Ion Torrent / PGM
3500DX/3730/3130/310
Increasing Read Length
“Large”
Genomes
“Microbial-sized”
Genomes
Increasing Throughput
Confirm &
Test
Resequencing Amplicons > 100bp on
PGM
• Most resequencing assays are based on PCR products
(amplicons) using Sanger sequencing chemistry and
capillary electrophoresis
• Amplicons designed for Sanger resequencing assays
are typically longer than current read length of PGM™
sequencer (100 bp)
• In the anticipation of rapidly increasing read lengths it
would be nice not to have to redesign the amplicons
• We hereby demonstrate how customers can sequence
any established PCR assay on PGM™ sequencer now
5
Confidential and Proprietary—DO NOT DUPLICATE
Detecting mutations of CFTR gene*
• The human Cystic Fibrosis Transmembrane
conductance Regulator (CFTR) protein is encoded by
the CFTR gene, ~200 kbp, 27 exons
• CFTR is a ABC transporter-class ion channel that
transports chloride and thiocyanate ions across
epithelial cell membranes
• Research on CFTR gene include detection of point
mutations or insertions and deletions.
• * some info from wikipedia
6
Confidential and Proprietary—DO NOT DUPLICATE
CFTR Amplicons Resequencing Workflow
A
Sample: Coriell gDNA
• Known mutations of CFTR gene
Amplification:
• MASTR CFTR kit; Multiplicon N.V.
• 34 amplicons; 2 multiplex PCRs
• 8,947 bases amplified
• Amplicons by MVZ genteQ GmbH
Sequencing: Ion 314™ Kit
• 2 hours
• 9.65 Mbp*
• 92,589 reads
• 104 bp average read length
•
7
* excluding primers from CFTR
MASTR kit
B
C
D
E
F
PCR on gDNA
Concatenation of Amplicons
Library Prep
Template Prep
Sequencing
2 hours
Analysis
Confidential and Proprietary—DO NOT DUPLICATE
Analysis
• PGM generated raw data generated
– Type: bases
– Format: fastaq
• Reference: CFTR gene sequence
– NCBI gi|287325315|ref|NG_016465.1|, 195703 bp
• Analysis 1: full data set
– Estimation of maximum coverage
• Analysis 2: subset of 4,001 reads only
– Suitable for multiplexing of samples within a single chip*
• Mapping: Targeted Resequencing pipeline of SeqMan®
NGen®, DNAStar Inc.
• Visualization: SeqMan® Pro, DNAStar Inc.
• * available Q2 2011
8
Confidential and Proprietary—DO NOT DUPLICATE
Coverage plots, UCSC Genome
Browser
• CFTR MASTR regions covered at 100%
• Typical coverage using full data set
9
Confidential and Proprietary—DO NOT DUPLICATE
Detection of Variants
• Evaluation of detection at low coverage (4,001 reads)
• 5 point mutations and 1 insertion/deletion were detected
–
–
–
–
–
–
c.869+11C>T
c.1408A>G
c.2562T>G
c.4389G>A
c.1-8G>C
c.1521_1523delCTT
• Mutation pattern allowed correct identification of the
blinded sample
10
Confidential and Proprietary—DO NOT DUPLICATE
Detection of Variants – cont.
c.869+11C>T
c.4389G>A
c.1521_1523delCTT
c.2562T>G
c.1408G>A
c.1-8G>C
11
Confidential and Proprietary—DO NOT DUPLICATE
Summary
• Concatenated amplicons can be used as template for
PGM™ system sequencing
• No need to redesign amplicons
• Target coverage is not impacted
• Point mutations and insertions/deletions can be detected
12
Confidential and Proprietary—DO NOT DUPLICATE
Life Technologies Products
•
•
•
•
•
•
•
•
•
•
13
T4 DNA Ligase
PureLink® PCR Purification Kit
Ion XpressTM Fragment Library Kit
Qubit® dsDNA HS Assay Kit
Qubit ® 2.0 Fluorometer
Ion OneTouch™ System
Ion Template 314™ Kit
Ion Sequencing 314™ Kit
Personal Genome Machine™ (PGM™) Sequencer
Torrent Server and Torrent Suite Software
Confidential and Proprietary—DO NOT DUPLICATE
About the project
• Scientific contributors. Thanks to:
– Dr Achmann, Dr Auber, genteQ, Hamburg, Germany
– Geoffrey Henno, Jurgen Del-Favero, Multiplicon N.V., Belgium
• This project was led by Alain Rico, FALCON team,
Europe, Life Technologies
• Acknowledgements
– Sarah O’Meara, Thierry Scarcez, Liqun He, Jason Myers (all Life
Technologies)
Legal
•
•
•
© 2011 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of
Life Technologies Corporation or their respective owner.
SeqMan and NGen are registered trademarks of DNASTAR, Inc.
For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
16
Confidential and Proprietary—DO NOT DUPLICATE
Appendix
17
Confidential and Proprietary—DO NOT DUPLICATE
My Lab
•
•
•
•
Ion Torrent required equipment*
DNA fragmentation device
Bioanalyzer
PCR workstation
Thermal cycler
Equipment common to next gen
sequencing
Note: In addition to equipment shown, an 18 mΩ water system is also required
18
Confidential and Proprietary—DO NOT DUPLICATE
What are the Life Grand Challenges?
• Seven, $1 Million Prizes
• First-of-its-kind
crowdsourcing initiative for
the Life Sciences
• Intended to promote
community growth around
open technologies
• Will accelerate
technology, innovation
and discovery
Confidential and Proprietary—DO NOT DUPLICATE
Three Ion Grand Challenges Announced
Speed Challenge – 2x Sample Prep Time
Scalability Challenge – 2x Bases
Accuracy Challenge – 2x Accuracy
$1M USD Prize for Each Challenge - $3M USD Total!
Confidential and Proprietary—DO NOT DUPLICATE
Grand Challenges Framework
Submission Window
3 months
Setting
the Bar
Accepting
Entries
Public
Challenge
Repeat as Necessary
Ion will continuously raise the bar !
Confidential and Proprietary—DO NOT DUPLICATE
Life Tech Pavilion
All ‘Life Grand Challenges’
housed in the Life Tech
Pavilion
Confidential and Proprietary—DO NOT DUPLICATE
Preconfigured Torrent Server
• Preloaded with Torrent Suite Software
• Converts raw signal to base calls
• Performs preliminary alignment to a reference
genome allowing run quality assessment
• Hosts web portal for data review and distribution
to collaborators
23
Confidential and Proprietary—DO NOT DUPLICATE
Torrent Server IT Service Contract
• Using Torrent Suite Software is as simple as using any web interface
• Keeping it updated and managing the data requires some Linux
experience.
• If you don’t have it, the BioTeam can help.
• Order it from the Ion Torrent Web Store.
Product
Part #
3 month support contract
4465993
12 month support contract
4465994
Confidential and Proprietary—DO NOT DUPLICATE
Torrent Pipeline – Data Summarization
Process Description
File Types
314 chip
316 chip
Raw Voltage Data
DAT
29 GB
141GB
Signal Processing
WELLS
1 GB
6 GB
Base Calls - Flow
SFF
0.5 GB
3 GB
Base Calls - Base
FASTQ
0.1 GB
0.6 GB
V1.3 File sizes for a single run of 65 cycles/260 flows enabling 120 base pair read
lengths.
Confidential and Proprietary—DO NOT DUPLICATE
How many runs can be stored?
Process
Equipment
314 / 1 PGM
316 / 1 PGM
Data Acquisition
PGM
50runs
10 runs
Raw Data
Torrent
Server
395runs
81runs
Archived Data
Torrent
Server
6000 runs
1200 runs
File sizes for a single run of 65 cycles that enables 120 base pair read length.
The Torrent Server has 12 TB of space with n=2 redundancy.
The above is not a guarantee or a specification. Simply to help the user guide usage.
Confidential and Proprietary—DO NOT DUPLICATE

similar documents