Ion Torrent

Ion Torrent : 个人化基因组测序仪
张远涛Ph.D
Sr Technical Sales Specialist
Yuantao.zhang@
主要内容：
Ion Torrent
Ion Torrent技术原理Nature文章
Ion Torrent: 个人化基因组测序仪
Main Frame
Sanger Sequencing Next‐Gen Sequencing Ion Semiconductor Sequencing
can use them.
使用方便的桌上型仪器
芯片既是测序仪
革命性的创新技术：
可升级的半导体技术+简单的化学反应原理
Semiconductor Manufacturing
H+
∆pH
Sensing Layer
Sensor Plate
Drain Source
Silicon Substrate
可升级的半导体技术–100X in the First Year
Ion 314™Chip Ion 316™Chip
Ion 318™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 runs
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Specification
简单的化学反应原理
简单的化学反应原理
Ion torrent ：整体工作流程
Clonal Amplification
DNA / RNA
Load Chip and Sequence
Ion Paired-end Sequencing（双向测序）
Innovative automated template preparation for PGM sequencer matches •Complete end-to-end workflow within 1 day or multiple samples per day
文库制备
模板制备
测序
3+hr
3 hr
1.5 hr
0.5 hr
快速：整个工作流程仅需1天时间
featuring low input material and fast and streamlined workflow
Analysis 3+ hours 2 hours
1 hour
Genetic diversity and population structure of the endangered marsupial Sarcophilus harrisii
Miller et al., PNAS (2011)
/animals/mammals/tasmanian-devil/
Rapid sequencing, de novo assembly & identification of novel microbial strains.
Rapid sequencing, de novo assembly & identification of novel microbial strains.德国大肠杆菌爆发: 用Ion torrent 三天内得到变异的大肠杆菌基因组序列
E coli 55989 complete genome
E coli LB226692 draft assembly
Monday May 30*Library preparation
O104:H4 and HUSC41 samples (reference) strain libraries prepared
Tuesday May 31Sequencing runs
0104:H4 amplified and sequenced 2 x 2 runs (Ion 314)Wednesday June 01 Sequencing runs
0104:H4 sequenced 3 x 2 runs (Ion 314)
Thursday June 02 Assembly
Draft Genome identified, Assembled, Submitted and Released from NCBI Friday June 03
Assay Design
TaqMan Assays Designed
*May 22 CEDC reports significant increase in patients with hemolytic uremic syndrome
“The biggest advantage [of the PGM] from my point of view as a public health official is that it's speedy, and speed is what is needed at the moment,”
Prof. Dr. Med Dag Harmsen, University Hospital Muenster
“[The PGM] takes the shortest time to generate genomic data.”Junjie Qin, BGI
荷兰肺炎杆菌爆发：Ion torrent再次大显身手
strain_1191100241
the RIVM
Enterohemorrhagic E. coli & Klebsiella Pneumoniae Outbreaks NEJM
Mark Pallen MD, Ph.D., Holger Rohde, M.D., Junjie Qin, Ph.D et al PLoS one
Alexander Mellmann., Dag
Harmsen*., Craig A. Cummings.,
et al
Science
Alexander Mellmann., Dag
Harmsen*., Craig A.
Cummings.
金黄色葡萄球菌抗药基因进化研究
美国马赛诸塞州综合医院：石蜡切片的体细胞突变检测（KRAS ）
Demonstrated the ability to detect somatic mutations to ~5% with accuracy of ~99.5%Single day workflow using fusion primer approach Ion DNA Barcoding Kit (Up to 16) available in Q3 for simultaneous analysis of multiple samples
KRAS exon 2
Mean per base error: 0.43%
26% freq. variant detection 575X Coverage
Dr. Long Le, Mass General Hospital
Prepare library via fusion primers or post-PCR ligation Fragment via mechanical or enzymatic shearing
Template Sequence Analysis
Library Amplicon sequencing using the fusion primer bidirectional sequencing approach for FFPE tumor tissue samples. A series of 10 amplicons were created to demonstrate concordance with Mass Gen’s current gold standard SNaPshot capillary sequencing based assay
Amplicon sequencing using the fusion primer bidirectional sequencing approach for FFPE tumor tissue samples. A series of 10 amplicons were created to demonstrate concordance with Mass Gen’s current gold standard SNaPshot capillary sequencing based assay
美国亚利桑那州肿瘤中心：体细胞突变验证（BRAF&NRAS ）
Demonstrated sequencing is possible from 6 year old FFPE tissue.
53 samples sequenced representing 47 unique samples on four 314 chips
Per 314 Chip: 14 barcoded samples x 5 amplicons/sample + 2 controls
Positive controls 100% accurate and Mutations found in 47% of samples
10 BRAF (V600E,K) and 12 NRAS codon 61 (Q61R,K,H)No sample had more than one mutation
Template Sequence Analysis
Library Barcoded amplicon sequencing for detection of BRAF & NRAS mutations in melanomas
Barcoded amplicon sequencing for detection of BRAF & NRAS mutations in melanomas 2 rounds of PCR using bar-coded primer sets allowed 16 samples x 5 amplicons per 314 chip (14 samples plus 2 controls)
Melanoma Cell Line A375
Positive Control
Melanoma Sample
GenBank Sample
Dr. George Watts
Deep sequence for heteroplasmy
detection ( > 1000x coverage on Ion 314)Ability to do 16 samples per run with barcoding
Accurate variant calling, especially in hypervariable regions of mitochondria
美国宾州州立大学：人类线粒体基因组测序（遗传疾病风险评估）
Mutation detected on position 15450
Prof/. Stefan Schuster Penn. State University
Amplify mtDNA via two overlapping long range PCR Fragment via mechanical or enzymatic shearing
Template
Sequence
Analysis
Library
Rapid resequencing of mitochondrial genomes for disease research, biodiversity assessment
Rapid resequencing of mitochondrial genomes for disease research, biodiversity assessment
澳大利亚西澳大学医学中心：人类线粒体基因组测序服务
AmpliSeq™肿瘤基因筛查芯片
AmpliSeq™肿瘤基因筛查芯片: 肿瘤基因组成
美国贝勒医学院推出AmpliSeq™肿瘤基因筛查芯片的检测服务
客户端：200bp 读长测序结果
• University of Münster • Broad Institute
Aligned Q20  (Mb) 380
200Q17 reads 1,912,390
Percent reads >200  bp 61%
Aligned Q20  (Mb) 323 
200Q17  reads 1,822,145
Percent reads >200  bp 62.2%
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318 Chip ：1.27G测序数据 和 391 bp平均读长


525 bp 读长
CGCTAAGTAATATTCGCCCCGTTCACACGATTCCTCTGTAGTTCAGTCGGTAGAACGGCGGACTGTTAATCCGTATGTCACTGGTTCGAGTCCAGTCAGA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CGCTAAGTAATATTCGCCCCGTTCACACGATTCCTCTGTAGTTCAGTCGGTAGAACGGCGGACTGTTAATCCGTATGTCACTGGTTCGAGTCCAGTCAGA 1 100 GGAGCCAAATTCTAAAAATTCGCTTTTTTAGCGCAATGTCACTGACCTTAGTTGAACATTGTTTTTTAACGGATAGCGGGTTTTTAACATCTTAAGCGCC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GGAGCCAAATTCTAAAAATTCGCTTTTTTAGCGCAATGTCACTGACCTTAGTTGAACATTGTTTTTTAACGGATAGCGGGTTTTTAACATCTTAAGCGCC 101 200 CTCGACCTTTATGGTTGAGGGCGTTTTGCTATGAACGCCATCACCATTTTCCCCTCGATTATAAAACTTGAGTTATTCAGTAGTCTCCCCTCTTGCAACT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CTCGACCTTTATGGTTGAGGGCGTTTTGCTATGAACGCCATCACCATTTTCCCCTCGATTATAAAACTTGAGTTATTCAGTAGTCTCCCCTCTTGCAACT 201 300 CACACCCAAAACTGCCTAACGAAAAGTTATTAATTTTCAATCATATTGCTATCAGTATTTACATTTTTTCGCTGTGCTAGAAAGGGCGCATTTATGTTAG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CACACCCAAAACTGCCTAACGAAAAGTTATTAATTTTCAATCATATTGCTATCAGTATTTACATTTTTTCGCTGTGCTAGAAAGGGCGCATTTATGTTAG 301 400 CTCGTTCAGGGAAGGTAAGCATGGCTACGAAGAAGAGAAGTGGAGAAGAAATAAATGACCGACAAATATTATGCGGGATGGGAATTAAACTACGCCGCTT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CTCGTTCAGGGAAGGTAAGCATGGCTACGAAGAAGAGAAGTGGAGAAGAAATAAATGACCGACAAATATTATGCGGGATGGGAATTAAACTACGCCGCTT 401 500 AACTGCGGGTATCTGTCTGATAACT ||||||||||||||||||||||||| AACTGCGGGTATCTGTCTGATAACT 501
600
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Ion Torrent 产品
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Personal Genome Machine™ (PGM™) Products
The Chip is the Machine™
Barcode Kit Library Kits (RNA and DNA) Control Kit TaqMan® Library Quant Kit Ion Sphere Control Kit Ion OneTouch™ Template Kit
Bioruptor
Ion Personal Genome Machine (PGM) Ion 314™ Chip (1M wells) Ion 316™ Chip (6M wells) Ion 318™ Chip (12M wells) Sequencing Kit
IKA
Torrent Server & Torrent Suite
QuBit® Fluorimeter
Argon Gas & 18MOhm Water
Some products have not yet been officially released and information about those products is subject to change without notice
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Ion Core Kits-100 and 200bp测序试剂盒


to-use reagents for quality control of every step
Ion对照试剂盒 for PGM™ Workflow
Ready-
Ion Control Kit • Library Control • Template Control • Sequencing Control
• •
Ion Sphere Quality Control Kit
• Pre- and post-enrichment quality control of Ion Spheres Ready-to-use reagents contain Fam™ and Cy-5® Dye labeled primers targeting Ion adaptors Uses Qubit® 2.0 Fluorometer
E.coli DH10b Control gDNA
E.coli DH10b Control Library
Control Ion Sphere™ Particles
Library
Template
Sequence
Analysis
Library
Template
Sequence
Analysis
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Ion Community ：Ion用户论坛

Community Access • User Videos • User Wiki • Application notes • FAQs
Customer Access • Site prep • User guides • Torrent Suite Guides • Release notes • File formats • Database schema
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PGM & the Ion OneTouch System
• Speed, Scalability and Simplicity • More reads • Longer reads • Higher quality • Higher throughput • Shorter time to result
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Thank For Your Time!!
Questions?

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