Lab methods: Exome / Genome 27 06 2013 Ewart de Bruijn
Library prep is only a small part of the complete DNA analysis workflow DNA isolation library prep enrichment flowchip prep sequencing bioinformatics interpretation PROBLEM All steps are very sensitive, linked to each other, and interchangeable by many details TREND Solutions are getting more and more boxed. Automation is more and more important. DIRECTION NGS more accessible but more and more a black box. (is that a good thing???...)
DNA isolation My advise 1 ug genomic DNA after two purifications with Qiagen Genomic tip 20/G (cat.no. 10223) Qiagen Dneasy Blood & Tissue kit (cat.no.69504) Use RNAse treatment Qbit for quantification Gel electroforese for quality assesment
Library prep (adapter ligation) makes a big difference but not between platforms Shear DNA End repair A tailing Adapter Ligation Nick translation Amplification Size selection Quantification normalization AMPURE XP Different than SOLID and Illumina, Ion Torrent s official protocol uses blunt end adapter ligation However, all adapter ligation reagent kits are inter-usable
Library preparation What is important (imo) High yield per cycle Low bias High fidelity Cost efficient time My advise Use a kit(s) that has separate buffers for (almost) every enzymatic step fragmentation by shearing Half your reagents for cost efficiency, use input range to see if depletion occurs Improving library prep is a possibility, test for yourself in your environment
Library preparation; A pitfall" % input Poor man s qpcr complexity
Adapters and primers FLt1 FLt2 SA1 SA2 SAt1 SAt2 After adapter ligation 123 123 133 130 120 128 After amplification (8x) 365 340 732 727 768 688 Yield (NG) FullLength-t ShortAdapters ShortAdapters-t Condition depending!! These results are in our lab environment. Other reagents or concentrations can influence yield
Ampure and sample prep buffer Cleaning up of a 50 bp marker in 1x reaction buffer conditions 0,5x 1x 2x 50 kb ladder 0,5x 1x 2x 3x 3x 100 bp fragments stay in a 2x ampure ratio cleanup
Automation (Biomek FXP) Automation is not difficult. Our deck-layout is easy and simple Sample prep is more repetitive and robust Supports easily 500 pg input Very low reaction volumes for cost effectiveness
Exome enrichments (custom available) Agilent SureSelect (XT/XT2) Agilent Haloplex Illumina TruSeq Exome Enrichment Kit Illumina Nextera Rapid Capture Exome Kit Roche SeqCap EZ Human Exome Library v3.0 Life Technologies TargetSeq Exome Enrichment We use SureSelect and follow the protocol to the letter. For one exception.. pooling
Scaling challenge Sample 1 Sample 2 Sample 3 Sample 4 Sample xxx Enrichment 1 Enrichment 2 Enrichment 3 Enrichment 4 Enrichment xxx Single sequencing run When target footprint becomes smaller, sample prep and enrichment bottlenecks becomes larger when aiming for a maximum benefit form NGS run capacity Solutions - Automation library and enrichment preparation - Multiplexing - Combined sample preparation and enrichment
Reduce work and costs by multiplexing Single-plex enrichment multiplex sequencing multiplex enrichment multiplex sequencing sample 1 sample 2 sample x sample 1 sample 2 sample x enrichment 1 enrichment 2 enrichment x add barcode add barcode multiplexing multiplexing enrichment sequencing 20 samples 20 enrichments 1 sequencing run Nijman et al, Nature Methods, 2010 Harakalova et al, Nature Protocols, 2011 sequencing 20 samples 1 enrichment 1 sequencing run
Whole genome sequencing Coverage bias is the biggest problem (DNA isolation, amplification and duplicates) Amplification free sample prep results in better evenness
An Example; Non-Invasive Trisomy Test Implement NITT in diagnostic environment Screening of at least 100 plus blood samples for trisomies 21, 18 and 13 Workflow 1 ml plasma (9-20 weeks pregnancy high risk) Custom sample prep Wildfire sequencing platform Z-score statistics based on sequence-tag counting Results ~50 samples done (35 bp, 10M U 0mm) 6 trisomies have been found correctly (blind study) untill now Future aims Automation. DNA isolation and sample prep overnight on a single platform Data analysis. Optimize bioinformatics. Testing multiple scripts. Include sex determination. 1 week turn around time. www.panoramatest.com
SOLiD Wildfire Technology Sequencing Simplify template prep workflow and reduce costs Emulsion PCR sample preparacon eliminated Sequencing beads (& bead- deposicon) eliminated Less- than 2 hours from library to sequencing directly on 5500 with <30 minutes hands on Cme! High density sequencing colonies à Over 600K
Two- sided Sequencing Further Enhances Throughput Colonies on top surface Flow-Cell objective 981,000 per mm2 FlowChip fluid channel Colonies on bottom surface 905,000 per mm2
Template Walking - Direct Library AmplificaCon in 5500 FlowChip Lane in a Single, ~ 30 Minute, Isothermal Step gdna Template hybridization Extension 37 C 3 min 1 copy of template Walking 60 C P2 (T) n (T) n (T) n (A) n 4 copies of templates P2 Extension (T) n Walking 2 copies of templates P2 Extension and strand displacement (A) n
Final remarks Sample prep is small part of the workflow Many options. All work. Ultimate method does not exist Agilent SureSelect is our method of choice WGS is better with amplification free sample prep Automation is key for robust, consistent and cost-effectiveness There is room for sample prep optimization! (NITT)
Thank you