次世代シーケンサの臨床応用
ライフテクノロジーズジャパン
片山 稔
1For Research Use Only. Not for use in diagnostic procedures.

Cost for Human genome sequence$ 2.7 Billion$1.5 Millon
Human GenomeProjectIon Proton™ II CHIP
660 million well
1 x Humam Genome>20 x Coverage
1,000$ /Run

$100,000SangarLaunch NGS(2005)(Launch mid 2013)$10,000$1,000

1990
22003

2008

2010

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

2013

次世代シーケンサが身近に™
Ion PGM
3

1Gbの、さらにその先へ

Ion Proton™

Gene to Genome by Next generation
sequencer“For genes”

4“For exome & genomes”

NGS potential as a diagnostic tool:

Coverage

43613843
Limitation:
15~20 % mutation
Variant coverage

Reference sequence

RefVariant

Var Freq

Coverage

Ref Cov

Var Cov

GT

59.66

24152

9739

14409

On sequence variant detection
NGS can get both of qualitative and quantitative data in same time!
AND low mutation can detect than Sanger sequence!!

Examples for NGS Application
Sequence application

RNA and DNA
sequence changes
DNA structure
variation

Methylation

RNA expression
profiles
6Application for neoplastic condition
Risk assessment
Risk management

Patient stratification
Predictions of therapeuticresponse

Micro-RNAs
Somatic/driver mutations

Prognosis

Methylation
Epigenetic changes
Molecular profiling

Therapeutic monitoring

Tumor sub-typing
Alterations in gene expression

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

Ion AmpliSeq™ Technology: As
Simple As PCR

Single-tube, ultra-high multiplex for Targeted Resequencing
Up to 3072 primer pairs+10ng DNA

Multiplex PCR: Maximum 3072 amplion from 1 tube reaction
PrimerA Pair

locusAlocusG

PrimerB Pair PrimerC PairlocusB

Construct Library7

3.5 hours

locusC

Prepare Template

PrimerD Pair

PrimerE Pair

PrimerF Pair

locusDlocusE

Run Sequence

Analyze Data

PrimerG Pair

locusFAmpliSeq™ panels

Cancer Primer
Pool46 genes
739 mutations

8Comprehensive
Cancer Panel
409 genes

Inherited Disease
Panel328 genes>700 diseases

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

Ion AmpliSeq™
Designerwww.ampliseq.com
24 to 3,072
amplicons/tube

Example for AmpliSeq Custom panel
Knight cancer institutehttp://www.knightdxlabs.com/home/search-results?q=ampliseq

Diagnostic Spectrum
NGS

SangerSingle geneTo
Several genes
Targeted
Gene/VariantAnalysis

Multi-Gene
PanelsWholeExome

WholeGenome

Multi gene to entire genome

Increasing Complexity

Clinical implementation of NGS• Division of Laboratory Science and Standards (CDC)• Genetic Testing Reference Material Coordination
Program (Get-RM) (CDC)
http://www.cdc.gov/dls/genetics/rmmaterials/default.aspx••••

Clinical Laboratory Standards Institute (CLSI)
American College of Medical Genetics (ACMG)
College of American Pathologists (CAP)
Association For Molecular Pathology (AMP)Clinical decision support systemsExample•Expert interpretation and guidance•Proficiency testing and alternative assessment are challenging
Ion Torrent PGM*

Genomic Analysis Interpretation

From sequences…

… to biologicalunderstanding…

…to actionable
information

Ion Semiconductor Sequencing
Rapid, Benchtop Sequencing for All

For more information, visit http://lifetechnologies.com/proton