DNA Services Overview November 20, 2012 1 © 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 2 Sequencing & Analysis Services at a glance WHAT WE DO NOT CURRENTLY OFFER . . . 3 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 • • • • • • • 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 4 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 5 …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 6 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 7 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 8 Library Preparation Guidelines We use either Illumina TruSeq or Agilent SureSelect for library prep Currently evaluating Nextera, but not yet in production We do not use NuGen kits Required for everything but sequencing of PCR products Whole genome resequencing Targeted resequencing RNA-Seq Minimal sample requirements (as per kit providers protocols) 1 µg purified DNA for TruSeq libraries 3 µg for purified DNA for SureSelect libraries Lower input amounts are at sample owners risk All incoming DNA samples subjected to QC Gel electrophoresis-qualitative assessment for degradation Picogreen fluorometry -quantitation 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 Any unused library can be returned to owner or destroyed Return requires charges for packaging and shipping 9 Targeted Capture Guidelines We currently use Agilent SureSelect products Whole exome Custom Kinome Others Can/will sequence captured materials sent to us TruSeq Exome Enrichment Nimblegen 10 Thank You! For additional information, please contact: dawn.casey@perkinelmer.com SeqSales@perkinelmer.com Phone: +1-877-737-5468 11