DNA Services Overview - Next Generation Sequencing

Report
DNA Services Overview
November 20, 2012
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© 2010
2009 PerkinElmer
Sequencing & Analysis Services at a glance
WHAT WE CAN DO . . .
 Illumina or Agilent library preparation
 Clustered flowcell generation
 Paired end and single end sequencing 2x100
 Whole genome
 Adapted libraries
 Prepared flowcells
 Target capture by hybridization
 Agilent SureSelect exomes
 Agilent SureSelect custom targeted
 Illumina TruSeq Amplicon-Cancer panel*
 Data Analysis
*available January 2013
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Sequencing & Analysis Services at a glance
WHAT WE DO NOT CURRENTLY OFFER . . .
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In house DNA or RNA extraction - Rutgers
In house RNA to cDNA conversion
Specialized/custom sample prep – chip / methyl
Long-read sequencing
De novo assembly – will allign to provided reference
Sequencing & Analysis Services at PKI
 Product Offering
High Throughput Sample Prep
• Automated library prep for sequencing
• Automated target capture by hybridization
•Strict SOP-driven processes
•Extensive quality checking
•CT licensed clinical lab (No. CL-0685)
•CLIA registered ( ID# 07D2034530)
• Project consulting services
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Sequencing Apps
Exome re-sequencing
Targeted re-sequencing
Microbial re-sequencing
Prepared library re-sequencing
Whole genome re-sequencing (on demand)
RNA
Clinical sequencing
Operational Infrastructure
• 4—6 week turnaround
•LIMS for sample tracking
• 3 HiSeqs + 1 MiSeq
• > 6000 samples successfully processed to date
• Secure private cloud in sequencing center; semi-public cloud hosted by PerkinElmer Informatics
•Current analysis capacity of 50-60 exomes per day—highly scaleable computing infrastructure
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Employing Proven Technologies & World-Class Automation…
PerkinElmer is focused on bringing best in class technologies –
we continually evaluate new technologies to ensure we are providing
the best available systems & processes to our customers
CURRENT PLATFORMS
SAMPLE & PROCESS
TRACKING
PerkinElmer
Informatics LIMS:
providing sample
tracking and status
updates
AUTOMATION
PKI JANUS
Automated
Workstations
PKI EnSpire
Plate Reader
TARGETED
CAPTURE
Agilent
SureSelect
SEQUENCING
Illumina HiSeq
2000 system
Illumina MiSeq
ANALYSIS
PerkinElmer
Informatics
Analysis Edition:
private cloud
implementation
Caliper Labchip
GXII
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…ensures faster turn-around-times and high quality control
Workflow flexibility to accommodate customer needs
Prepared by us
•Libraries
•Captures
•Flowcells
DNA Library
preparation
Target
Enrichment
[Optional]
Automated
Amplification
(Clustering)
Prepared by customer
•Libraries
•Captures
•Flowcells
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Automated
Sequencing
Automated
Data
Analysis
Illumina HiSeq 2000-Overview
Variable read lengths (50-100bp)
Sequence in one or two directions
Up to 2.8 x109 reads per flow cell (280 Gb)
Run time is variable
 1 x 50bp =~ 2.5 days
 1 x 100bp = ~6 days
 2 x 100bp = ~12 days
Output deliverables can vary depending on project
requirements
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Sample Submission
Each sample submission needs to be accompanied by sample manifest
 Required DNA concentration is ≥25 ng/µL
 Required concentration for ready-to-sequence libraries is ≥ 10nM
 Required amount of SureSelect XT library for capture ≥ 700ng
When submitting, send shipping notification to Seqproject @ perkinelmer.com along with
electronic copy of shipping manifest and tracking number
Samples should be shipped overnight via express shipping
Follow instructions contained in “Sample Prep and Shipping Guidelines_v3.2” for sample
purity, concentration, etc.
Not following instructions will delay the project
Include hardcopy of sample manifest with samples
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Library Preparation Guidelines
We use either Illumina TruSeq or Agilent SureSelect for library prep
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Currently evaluating Nextera, but not yet in production
We do not use NuGen kits
Required for everything but sequencing of PCR products
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Whole genome resequencing
Targeted resequencing
RNA-Seq
Minimal sample requirements (as per kit providers protocols)
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1 µg purified DNA for TruSeq libraries
3 µg for purified DNA for SureSelect libraries
 Lower input amounts are at sample owners risk
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All incoming DNA samples subjected to QC
 Gel electrophoresis-qualitative assessment for degradation
 Picogreen fluorometry -quantitation
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Samples failing QC will not be processed without consent of owner
Any unused sample can be returned to owner or destroyed
 Return requires charges for packaging and shipping
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Any unused library can be returned to owner or destroyed
 Return requires charges for packaging and shipping
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Targeted Capture Guidelines
We currently use Agilent SureSelect products
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Whole exome
Custom
Kinome
Others
Can/will sequence captured materials sent to us
 TruSeq Exome Enrichment
 Nimblegen
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Thank You!
For additional information, please contact:
[email protected]
[email protected]
Phone: +1-877-737-5468
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