22 Sequencing & Analysis Services at a glance 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 WHAT WE CAN DO...
33 Sequencing & Analysis Services at a glance 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 WHAT WE DO NOT CURRENTLY OFFER...
44 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
55 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 …ensures faster turn-around-times and high quality control Employing Proven Technologies & World-Class Automation… SAMPLE & PROCESS TRACKING PerkinElmer Informatics LIMS: providing sample tracking and status updates CURRENT PLATFORMS PKI JANUS Automated Workstations PKI EnSpire Plate Reader Caliper Labchip GXII AUTOMATION Agilent SureSelect TARGETED CAPTURE Illumina HiSeq 2000 system Illumina MiSeq SEQUENCING PerkinElmer Informatics Analysis Edition: private cloud implementation ANALYSIS
66 DNA Library preparation Target Enrichment [Optional] Automated Amplification (Clustering) Automated Sequencing Automated Data Analysis Workflow flexibility to accommodate customer needs Prepared by us Libraries Captures Flowcells Prepared by customer Libraries Captures Flowcells
77 Variable read lengths ( 50-100bp ) Sequence in one or two directions Up to 2.8 x10 9 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 Illumina HiSeq 2000-Overview
88 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
99 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 Library Preparation Guidelines
10 We currently use Agilent SureSelect products Whole exome Custom Kinome Others Can/will sequence captured materials sent to us TruSeq Exome Enrichment Nimblegen Targeted Capture Guidelines