PerkinElmer Solutions for NGS
sample preparation
Paul Butler
World of Technology and Science, Utrecht
1
© 2009
2011 PerkinElmer
The evolution of DNA Sequencing (before Next Gen)
Slab Gel
Auto Radiography
3
Capillary Sequencing
Fluorescent
How many of these
=
throughput of one of these
60,000
4
6
1X10
9
60X10
Bases per day
Bases per day
Overview of PerkinElmer portfolio of genomic and NGS Solutions
Our complete portfolio of sample prep solutions upstream of
sequencing-Low, medium and high throughput
New product for library QC
LabChip GX Touch 24
Our dedicated NGS post sale support
Our library prep automation offer
Any chemistry and all sequencer platforms
Wide range of library prep protocols
Future development roadmap
Protocols validated via proof data from the sequencer and documentation
Our user community-over 180 automated suites in use for NGS
5
PerkinElmer Product Portfolio for NGS Workflows
LIMS – Informatics - Visualization
Isolate
Sample
Fragment
& Sizing
Generate
Library
Quantify
Sample
Sequence
Analyze
Prepito / chemagic 360 / JANUS chemagic • Nucleic Acid Extraction
LabChip XT • Automated DNA Fractionation and Sizing
LabChip GX Touch • Bioanalyzer-like QC for DNA and
RNA
Sciclone NGS Workstation •
Automated High-throughput Sequencing
Sample Preparation
Zephyr NGS Workstation
• Post PCR Workstation &
Sample Preparation
NGS Express • Bench6 Research Use Only. Not For Use in Diagnostic Procedures
For
top Sequencer Sample
Preparation
PerkinElmer loves the Agilent 2100 Bioanalyzer but………!
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New product -LabChip GX Touch 24 System for fragment size QC
System features:
Quantification & size QC of NGS libraries,
fragments and PCR products
Same chip technology as Bioanalyzer with
significantly lower cost per sample
New 1-24 sample batch size option
Multiple assay kits supporting analysis of 25
bp to 12kb fragments including Genomic DNA
integrity
Fast- 30-60 seconds per sample
Walk-away automation of sample batches of
1-24, 1-96 and 1-384
8 Research Use Only. Not For Use in Diagnostic Procedures
For
Full Featured Automation
LabChip GX Touch Process Flow
Steps:
Prepare the Chip
Load the Chip
Insert Sample Plate
Run Automated Assay
View Results in Minutes
The logical next step
for Bioanalyser users!
9 Research Use Only. Not For Use in Diagnostic Procedures
For
Enhanced Ease of Use
Genomic DNA quality-Egrams of Degraded vs. Intact gDNA
Intact
λ-DNA
Sample 2
Sample 3
Sample 4
Degraded
10
For Research Use Only. Not For Use in Diagnostic Procedures
10
Sample 5
Example GQS Scores – Scale from 0 (degraded) to 5 (intact)
Highest score: 4.1 (λ-DNA)
Lowest score: 1.0 (Sample 5)
4.1
λ-DNA
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For Research Use Only. Not For Use in Diagnostic Procedures
11
3.7
Sample 2
2.0
Sample 3
2.0
Sample 4
1.0
Sample 5
Sequencing Sample Prep Automation
NGS Express
Zephyr NGS
Sciclone NGSx
Sample
Capacity
Up to 24 samples per day
8 to 96 samples per day
3 to 5 batches each with 8 to 96
samples per week (depending on
application)
Key
Applications
- Library Prep (MiSeq, PGM, GS FLX)
- DNA normalization and pooling
- Ion Amplicon Prep
- MiSeq Nextera and Amplicon Prep
Post PCR
- SPRI bead purification
- Normalization & qPCR setup
- Library Prep (DNA, RNA)
- Targeted resequencing
- Library preparation (DNA, RNA)
- Sequence capture
- Normalization & pooling
- Custom protocols
Ideally for...
Labs with small bench top NGS systems
and moderate weekly thoughput
Workflows requiring seperation
of pre and post PCR
processes or for medium/low
throughput library prep
HT sample preparation (e.g. For
HiSeq 2000)
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LOW
Throughput Libraries/Application
HIGH
Informatics/integromics
Informatics/
integromics and LIMS Sample Tracking
Isolate
Sample
Fragment
& Sizing
Generate
Library
Quantify
Sample
Sequence
Analyze
Sequencers, applications, and chemistries: so many choices
And the list keeps growing…Don’t get locked in
Automation of Illumina chemistries
14
Automation of Illumina chemistries
User guides
Nextera Library prep
TruSeq stranded RNA
TruSeq DNA
TruSeq RNA
Nextera on NGS Express
Future applications development plan
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Automation of nearly all chemistries!
Ion Torrent AmpliSeq
PacBio
Kapa HTP DNA
NimbleGen SeqCap EZ
Agilent SureSelectXT
NEB
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Automated library preparation system for Ion
AmpliSeq™ technology by NGS Express™
LifeTechnologies Japan and Perkin Elmer Japan
Introduction
Ion AmpliSeq™ technology is a simple, efficient and rapid process for
enriching hundreds to thousands of genomic targets for next generation
sequencing from 10 ng of FFPE or whole genomic DNA in a single tube.
The flexible nature of this technology scales to meet the demands of both
basic and translational research. Although library preparation by manual
handling is acceptable for small scale, the trend towards larger
sequencing projects involving hundreds of samples presents a unique set
of challenges with respect to throughput, cost and uniformity. These
problems are compounded when involving associated processes such as
sample multiplexing. To avoid errors in library preparation lead to loss of
precious samples and wasted reagents. Consider automation to achieve
more uniform library preparations. With automation, it can increase the
quality of NGS libraries and lower overall costs, while eliminating tedious
repetitive procedures.
NGS Express™ Automated Liquid Handling Workstation is a compact,
flexible and easy-to-use library prep system for benchtop sequencers
such as the Ion Torrent PGM™ system. It is designed for efficient sample
preparation of up to 24 NGS libraries, and eliminates the need for timeconsuming protocol development.
Now we are development for automated library preparation for Ion
AmpliSeq™ technology by NGS Express™ Automated Liquid Handling
Workstation.
This systems will accelerate sample throughput for Ion AmpliSeq™
technology without handling error.
Ion AmpliSeq™ Technology
Result
Specification
Sequence summary
Targets
Up to 5Mb of just your genes or genomic regions
Amplicon length
150 bp (optimized for FFPE) or 200 bp (standard)
amplicons
Primer pool size
24 to 6144 primer pairs per pool
Input DNA required
10 ng per primer pool
Total base: 422M, Total reads: 3,618,544
Sample
name
Total
aligned
base toNA17828
hg19: 415M NA12762
Replicate number
Mapped Reads
Coverage at 1x
Coverage at 100x
Coverage at 500x
1
439,376
100.00%
98.86%
93.22%
2
431,569
100.00%
99.61%
97.35%
1
438,768
100.00%
99.61%
96.72%
2
408,173
100.00%
99.08%
95.99%
Uniformity of coverage
97.01%
98.81%
97.43%
97.10%
95.51%
97.64%
SK-MEL-28
1
2
439,554
432,692
100.00%
100.00%
98.86%
98.51%
95.71%
92.54%
97.09%
96.55%
MDA-MB-231 has deleted part of CDKN2A gene (c.1_471del471, COSMIC ID: COSS934536). Two
amplicons in Cancer HP v2 failed to amplify.
Workflow for Ion AmpliSeq™
Sample to variants in a single day
Work with NGS Express™
Confirm with known mutation
MDA-MB-231
+
NGS Express™ Workstation
Gene
AA Mutation
CDS Mutation
TP53
BRAF
KRAS
p.R280K
p.G464V
p.G13D
c.839G>A
c.1391G>T
c.38G>A
Gene
AA Mutation
CDS Mutation
BRAF
EGFR
p.V600E
p.P753S
c.1799T>A
c.2257C>T
Muation rate
1st
54.32
99.9
63.47
2nd
57.2
99.81
61.93
SK-MEL-28
Muation rate
1st
99.88
99.66
2nd
99.72
99.39
Comparison of mutation rate between replicate
Material and Method
Samples
Reference control – NA12878, NA12762
Cancel cell – MDA-MB-231, SK-MEL-28 (NCI-60 cell line)
Name
Tissue
Number of Known Mutation In Cancer HP v2
MDA-MB-231
Breast
3
Skin
2
SK-MEL-28
Primer pool
Ion AmpliSeq™ Cancer Hotspot Panel v2
Runs
8 barcoded run by 318 CHIP
Each sample run duplicate
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MDA-MB-231
1
2
483,895
457,308
98.84%
98.84%
97.93%
98.45%
94.43%
94.94%
For Research Use Only. Not intended for any animal or human therapeutic or
diagnostic use.
© 2013 Life Technologies Corporation / © 2013 PerkinElmer Japan Co., Ltd. All rights
reserved.
N = 47
Conclusion
We are successful to develop the automated library preparation system
for Ion AmpliSeq™ technology by NGS Express™ Automated Liquid
Handling Workstation. An automated protocol for preparing Ion AmpliSeq™
has been validated for production use. This system up to 24 libraries can be
prepared in a single day.
The result of mutation profile show high concordance in duplicate samples.
It shows this system has enough reproducibility for ultra-multiplex amplicon
re-sequence.
This system can reduce steps for manual handling on library preparation
step. Sequence data can get stable without any complicate procedure.
NGS Application Roadmap: NGS Express(1-24 libraries)
Vendor
Reagent Kit Name
Application Area
Completion Status
Beckman Coulter
SPRI Purification
Miscellaneous
Standard, Factory Installed
Beckman Coulter
SPRI Size Selection
Miscellaneous
Standard, Factory Installed
PerkinElmer
Normalization & Pooling
Miscellaneous
Standard, Factory Installed
Agilent
HaloPlex
Exome/Targeted Capture
Option, Field Installable
Agilent
SureSelect XT2
Exome/Targeted Capture
Option, Field Installable
Agilent
SureSelect Strand-Specific RNA Library Prep
RNA Library Preparation
Option, Field Installable
BiooScientific
NEXTflex DNA Sequencing
DNA Library Preparation
Option, Field Installable
Illumina
Nextera
DNA Library Preparation
Gold Standard
Illumina
Nextera Rapid Capture
Exome/Targeted Capture
Option, Field Installable
Illumina
Nextera XT
DNA Library Preparation
Option, Field Installable
Illumina
TruSeq Amplicon
Exome/Targeted Capture
Option, Field Installable
Illumina
TruSeq Nano
DNA Library Preparation
Option, Field Installable
Illumina
TruSeq PCR-Free
DNA Library Preparation
Option, Field Installable
Illumina
TruSeq RNA
RNA Library Preparation
Option, Field Installable
Illumina
TruSight Tumor
Exome/Targeted Capture
Option, Field Installable
KAPA Biosystems
SYBR FAST qPCR Kit
Quantification
Gold Standard
KAPA Biosystems
HTP Library preparation
DNA Library Preparation
Option, Field Installable
Life Technologies
(Ion Torrent) Ampliseq Panels
Exome/Targeted Capture
Gold Standard
Life Technologies
(Ion Torrent) Ion Total RNA Kit v2.0
RNA Library Preparation
Option, Field Installable (1-12)
New England BioLabs Inc.
NEBNext Ultra
DNA Library Preparation
Option, Field Installable
Roche NimbleGen
Nimblegen SeqCap EZ
Exome/Targeted Capture
Option, Field Installable
Pacific BioSciences
2kb Template Preparation & Sequencing
DNA Library Preparation
Option, Field Installable
Pacific BioSciences
10kb Template Preparation & Sequencing
DNA Library Preparation
Option, Field Installable
KAPA Biosystems
Hyper Prep Kit
DNA Library Preparation
In development
Life Technologies
(Invitrogen) PicoGreen
Quantification
In development
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NGS Roadmap: Sciclone NGS/NGSx(1-96 libraries)
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Application Area
DNA Library Preparation
DNA Library Preparation
DNA Library Preparation
DNA Library Preparation
DNA Library Preparation
DNA Library Preparation
DNA Library Preparation
DNA Library Preparation
DNA Library Preparation
DNA Library Preparation
DNA Library Preparation
DNA Library Preparation
Epigenetic Research
Exome/Targeted Capture
Exome/Targeted Capture
Exome/Targeted Capture
Exome/Targeted Capture
Exome/Targeted Capture
Exome/Targeted Capture
Quantification
Quantification
RNAseq
RNAseq
RNAseq
RNAseq
RNAseq
RNAseq
Varied
Varied
Varied
Reagent Kit Name
Illumina TruSeq DNA
Kapa Bio HTP Library Prep
PacBio DNA Library (10kb)
Illumina Nextera XT
NEBNext
PacBio DNA Library (2kb)
Illumina Nextera
Ion Torrent Fragment Library Prep
Nextera Rapid Capture
Illumina TruSeq PCR Free
NuGen Encore
Illumina TruSeq Nano
Custom Chromatin IP
Agilent SureSelect XT
Nimblegen SeqCapEZ User Guide.pdf
Illumina TruSeq Exome
Agilent HaloPlex
Agilent SureSelect XT2
Ion Torrent Ampliseq
KAPA Bio qPCR
PicoGreen Quantitation
Illumina TruSeq RNA
Illumina TruSeq Stranded Total RNA (Ribozero)
Illumina Stranded mRNA (poly-A selection)
Epicentre Ribozero/Scriptseq Stranded RNA
Illumina Truseq Small RNA
NuGen Ovation
SPRI Purification
SPRI Size Selection
Normalization & Pooling
Sciclone NGS/NGSx
Gold Standard (8-96): NOTE ILLUMINA HAS DISCONTINUED
Gold Standard (8-96)
Gold Standard (8-96)
Factory Installable (8-96)
Factory Installable (8-96)
Factory Installable (8-96)
Factory Installable (8-96)
Factory Installable (8-96)
Factory Installable (8-96)
Field Verified (8-96)
3rd Party Installed (8-96)
In Development (8-96)
Factory Installable (8-96)
Gold Standard (8-96)
Gold Standard (8-96)
Factory Installable (8-96): NOTE ILLUMINA HAS DISCONTINUED
Factory Installable (8-96)
Factory Installable (8-96)
In Development (8-96)
Factory Installable (8-32)
Factory Installable (8-96)
Gold Standard (8-96)
Gold Standard (8-96)
Factory Installable (8-96)
Field Verified (8-96)
Field Verified (8-48)
3rd Party Installed (8-96)
Factory Installable (8-96)
Factory Installable (8-96)
Factory Installable (8-96)
NGS Roadmap: Zephyr NGS
Application Area
Quantification
Varied
Varied
DNA Library Preparation
DNA Library Preparation
DNA Library Preparation
DNA Library Preparation
Exome/Targeted Capture
Quantification
Exome/Targeted Capture
Exome/Targeted Capture
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Reagent Kit Name
KAPA Bio qPCR
SPRI Purification
Normalization & Pooling
Illumina TruSeq DNA
NEBNext Ultra DNA Library Prep
KAPA HTP
NuGEN Ovation Ultralow Library Prep
Illumina TruSeq Amplicon
PicoGreen Quantitation
Agilent SureSelect XT
Ion Torrent Ampliseq v2.0
Zephyr NGS
Factory Installable (8-32)
Factory Installable (8-96)
Factory Installable (8-96)
Factory Installable (8-48)
Field Verified (8-48),
In Development (8-48)
Field Verified (8-48),
Factory Installable (8-96)
Factory Installable (8-96)
Field Verified (8-48),
In Development (8-48)
PerkinElmer NGS field support resources available in Europe
Several general automation specialists
3 Micro-fluidic technical specialistsVeronika Delcheva
Leslie Huber
Ilse Klein
3 dedicated NGS post sale automation specialists
Yuri Yakushko
Julien Rouzade
Jose Couto
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Automated FFPE DNA Extraction + Genomic DNA Analysis on GX AGBT’13
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Micro-RNA’s using LabChip XT-University of Miami
University of Miami
Tomas Westerling, Dana Farber Cancer Institute Boston
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Tomas Westerling, Dana Farber Cancer Institute Boston
A good sample result is where “PGR enhancer1” is high vs “DNA yield ng”
Sciclone automated protocol gives best reproducibility + highest PGR enhancer 1 vs DNA
yield
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PerkinElmer’s Solution to Genomics analysis: OmicsOffice
An Integrative Analysis Suite for Transcriptomics data
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So who uses our solutions?
OICR
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Massachusetts General Hospital
OICR
University of Queensland
UMC Groningen
Erasmus Rotterdam
RINEN
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Tokyo University
Edge BioSystems
Children’s Mercy
Hospital of
Kansas City *2
BGI Shenzen
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University of Miami
Johns Hopkins University
Yale University
Washington University *2
NCGR
JGI
Los Alamos National Lab
NIH (NHLBI)
MD Anderson Cancer Center
University of Maryland
Vanderbilt University
Hudson Alpha Institute
University of Connecticut Health Center
University of Iowa
University of Carolina
USAMRID
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The Genome Analysis Centre TGAC Norwich
Sequenom
Prince Wales Hospital, Chinese University in Hong
Kong
ELIM Biopharm
Complete Genomics
Nestle
Sanger Centre
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Dr Manabu Fujie, Ph.D, OIST, Okinawa, Japan
Applications and assays
Sciclone NGS Workstation running “home
brewed” exome capture library prep chemistries
Research
LabChip GX for library QC
To understand better the molecular mechanisms of coral
biology including decoding the approximately 420-megabase
genome of Acropora digitifera using NGS technology. Using
Illumina HiSeqs , Roche 454s, and Life Technologies Solid
34
Dr Belinda Whittle and Yafei Zhang, APF, ANU Canberra
Applications and assays
Research
The Australian Phenomics Facility (APF) is a specialised
facility for developing, characterising and archiving mouse
models of human disease, in particular Human and mouse
exome sequencing
NGS Express Workstation for processing of
enriched DNA libraries for their exome
sequencing pipeline.
LabChip GX for library QC
Back to Index
Account Manager: Christine Mylo
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Dr Gao Yuan,JHU Biomedical Engineering Faculty, Baltimore, USA
Applications and assays
Research
The genetic and epigenetic basis of brain function and
neurological diseases — with a focus on schizophrenia,
bipolar spectrum disorder and major depression. Nextgeneration sequencing is bringing the dream closer to the
reality of transforming biology into quantitative information.
Study brain transcriptome during development, using postmortem human brain and derived iPS cells science. Use
HiSeq2000 and 454 Junior
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Account Manager: Eric Herr
Sciclone NGS Workstation running exome
capture library prep
Zephyr
LabChip GX for library QC
LabChip XT
Back to Index
Toumy Guettouche, Ph.D. University of Miami Hussman Institute
Applications and assays
Research
The Center’s ultimate aim is to provide the necessary
technical tools and methodological “know-how” to enable a
complete systems approach to the study and analysis of
human disorders. Such studies include disease gene
localization (genotyping), gene expression analysis, modern
ultra-high-throughput next generation (NGS) sequencing and
re-sequencing, and analysis of the effects of epigenetic
abnormalities.
.
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Sciclone and Zephyr Workstations for
• Targeted re-sequencing
• Whole exome sequencing (also in CLIA lab)
• Whole genome sequencing
• RNAseq (all applications)
• ChipSeq
• TruSeq DNA and exome
• Agilent SureSelect XT
LabChip GX for library QC using gDNA assay
LabChip XT for miRNA’s
Back to Index
Account Manager: Eric Herr
Example: Detecting Rare Childhood Diseases
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Neonatal Diagnosis by WGS in Two Days!: STAT-Seq
Sci Tranl Med 4, 154ra135 (20120)
“ These kinds of innovation will help more hospitals
bring sequencing into clinical care, says Richard Gibbs,
director of the human genome sequencing centre at
Baylor College of Medicine in Houston, Texas. “A lot of
people are going to realize from this that the future is
now.”
Facilitated by debottlenecking sample prep and informatics
Zephyr NGS: Customer Testimonial
Dr. Michael A. Quail
Sequencing R&D Team Leader
The Wellcome Trust Sanger Institute, Hinxton/UK
“We find the Zephyr NGS easy to use and flexible enabling processing of
just one column of samples up to a whole plate. We mostly use it for
AMPure purification of post PCR NGS fragment libraries which are
recovered in high yield when compared to performing the protocol
manually.”
Capabilities
•
•
•
•
•
40
SPRI purification protocol
Normalization protocol
Pooling Protocol
qPCR set up
Custom methods
Zephyr NGS Workstation
Sciclone NGS: Customer Testimonial
Dr. Keith Harshman
Coordinator, Genomic Technologies Facility
Center for Integrative Genomics
University of Lausanne
“PerkinElmer’s combination of integrated hardware and software coupled with
their excellent technical support made it possible for us to increase our exome
library preparation output 10-fold”
Capabilities
•
•
•
•
41
Library preparation (DNA, RNA)
Sequence Capture
Normalization & Pooling
Custom methods
Sciclone NGS Workstation
NGS Automation Suite: Customer Testimonial
Jakob Hedegaard
M.Sc., Ph.D., Associate Professor
Department of Molecular Medicine (MOMA)
Aarhus University Hospital, Skejby, Denmark
“Using the Sciclone NGS, Zephyr NGS and the LabChip GX for our preparation of NGS
libraries, has resulted in higher throughput and reproducibility compared to manual library
preparation. We find the instruments easy to use and the supplied methods to be flexible
in terms of number of samples to process, volumes etc. We have received excellent
service and support from PerkinElmer, both during installation and operation.“
Pre-PCR
Sciclone NGS Workstation
42
DNA-Capture-Seq
- TruSeq DNA library preparation
- Pre-amplification and purification
- Pooling for Nimblegen capture
- Setting up post-capture PCR
RNA-Seq
- RiboZero rRNA depletion
- ScriptSeq library preparation
- Setting up amplification reactions
Post-PCR
Zephyr NGS Workstation
LabChip GX
- Purification of amplified libraries
- Library QC
- KAPA qPCR
- Normalization and pooling for RNA-Seq
- Custom methods
Publication by Sequenom (2013)
The development presented
here was preceeded by
research activities and followed
by additional verification and
validation studies conducted in
a CLIA-certified laboratory.
The combined semi-automated process produced 96 libraries in 5 hours,
requiring only a single technician and 1.5 hours of hands-on labor time.
This resulted in a 4-fold increase in throughput coincident with a 4-fold
decrease in labor without sacrificing library yield or quality.
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057381
43
44
KEC Case Study – Perkin Elmer
TGAC is the European Reference Site for Perkin Elmer
• Suite of Perkin Elmer instrumentation
• Gold Standard protocols
• Bespoke methods
• Enhanced programming
• Training workshops
• Visits and Tours
•
45
Knowledge Exchange and
Commercialisation
The Genome
Analysis
The Genome
Analysis Centre
Centre
The Perkin Elmer Suite at TGAC
LabChip GX
Zephyr with Twister 2
3 x Sciclone NGS (1 with
Twister 2 and MeCour cooling
tower)
NGS Express workstation
46
The Genome Analysis Centre
LabChip XT
Application
Current automated protocols carried out on Perkin Elmer
instrumentation at TGAC:
47
•
•
•
•
Illumina Truseq RNA
Illumina Truseq DNA
Illumina Nextera XT
NimbleGen SeqCap EZ Target Enrichment
•
•
•
•
Bead based size-selection
Library normalisation
Library pooling
Quality Control assay set up
The Genome Analysis Centre
BGI Shenzen China LabChip GX for NGS QC
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6 LabChip GX installed in BGI China, more worldwide
Broad Institute: Pico DNA Assay on GX for NGS QC
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51