Large scale plant genomics: breaking the sample preparation barrier Dr Adam James Carroll Australian National University Professor Murray Badger Lab The sequencing cost barrier has been broken Sample preparation is the new barrier! 35000 30000 25000 20000 15000 10000 5000 0 Sample Preparation Cost Sequencing Cost Sample Preparation Cost (%) 1 101 201 301 401 501 601 701 801 901 Number of Multiplexed Samples 100 90 80 70 60 50 40 30 20 10 0 % Sample Preparation Total Cost ($) Sample Preparation = $30 per sample Can we break the sample prep barrier? $3 per sample – is it possible? 6000 Sequencing Cost 5000 Sample Preparation Cost (%) 4000 3000 2000 1000 0 1 101 201 301 401 501 601 701 801 901 Number of Multiplexed Samples 100 90 80 70 60 50 40 30 20 10 0 % Sample Preparation Total Cost ($) Sample Preparation Cost Sample prep: broken down 1. Tissue collection and annotation 2. Tissue grinding / aliquoting 3. DNA/RNA extraction 4. Extract QC 5. Shearing 6. Library preparation 7. Library QC 8. Sequencing Sample Prep Service Providers DNA DNA Library Extraction Prep Min. $1.20 Max. $150 Avg. $30 Bioanalyzer DNA QC $40 $3 $1015 $125 $379 $32 Minimum sample prep charge (DNA NGS) = 1.20 + 40 + 3 = $44.20 per sample Sample Prep Service Providers Sample Collection RNA Extraction Bioanalyzer RNA QC RNA Library Prep Bioanalyzer DNA QC ? $10 $10 $150 $3 Max. ? $150 $12 $700 $125 Avg. $50 $11 $348 $32 Min. ? Minimum sample prep charge (RNA NGS) = 10 + 10 + 150 + 3 = $173 per sample Genomic Technologies Facility: Iowa State University Plant Sciences Institute Excellent services at amazing prices! http://www.plantgenomics.iastate.edu/fees.php Australian National Robotics Facility Tissue collection and annotation Collecting tissues introduces hidden costs: • Tube • Label • Metadata capture • Freezer storage • Sample tracking Mobile collection hardware/software Getac F110 Military Windows Tablet: • GPS • 1D Barcode reader • Waterproof • Heatproof • Drop proof • Hands-free harness Mobile collection hardware/software Accelerated collection: 1. Scan barcode (no labeling) 2. Location/time automatic 3. Push to buttons to select metadata 4. Freeze sample 5. Export metadata downstream Tissue grinding & dispensing: Labman / Max-Planck Cryogenic Grinder / Dispenser Grinds and weighs aliquots of frozen tissue Weighs out 5 to 100 mg +/- 0.5 mg Will output up to 500 samples/day Cost approx. $USD300k Automated Sample Preparation Perkin Elmer Janus Automated Workstation Will automate: • Liquid handling • Capping / decapping of screw caps • Centrifugation • Heating/Agitating • Freezing • Magnetic bead steps • Automated DNA/RNA extraction, quantitation, normalisation and library prep! Reagent dispensing Liquid level detection Gripper arm and PlateStack Integrated FluidX XSD96-Pro Capper/Decapper Integrated Hettich robotic centrifuge CTAB + MagAttract DNA Extraction • Cheap • Suited for automation • Good performance with difficult species Xin and Chen (2012) Plant Methods 8:26 CTAB + MagAttract DNA Extraction Modified CTAB Extraction: • Introduced chloroform extraction step - increased yield • Introduced modified MagAttract bead cleanup step • Increased yield 30-fold • Reduced MagAttract beads 4-fold ($0.1 / sample) • Achieved high purity • Worked well with ‘difficult’ pine needles Xin and Chen (2012) Plant Methods 8:26 So how cheaply can we automatically extract 96 DNA samples? 192 Tubes $27 1000 tips $63 MagAttract Beads $9 2 Microtitre plates $18 $US 1.20 per sample In one day: 20 x 96 samples, saving 50 hours of labour. In one year: 365 days * 50 hours * $30 / hour = $547,500 = 1,806,750 RM DNA/RNA Quant and Purity Check: Caliper LabChip DS multi-channel microfluidic UV/Vis plate reader Specifications:  Sensitivity to 5 ng/ul for DNA  Dynamic range to 5500 ng/ul  DNA Quantification  5 minutes per 96 well plate  Fits automated workflow  Reduce DNA library amplification artifacts DropPlate 96: Disposable 96-well µl-microtiterplate DNA/RNA/Protein Size Profiling: Caliper LabChip GXII High-throughput size profiling of DNA, RNA and protein mixtures Approx $1 ± 0.4 / sample Approx 100 samples / hour Extract and Library QC: Caliper LabChip GXII NGS Express – Automated NGS sample prep System Includes 1 – 24 Samples 4 – Tip VariSpan with 250 µL syringes JANUS Application Assistant GUI Heating/Cooling Stations and Shaker Location Built in Protocols for  PCR Setup  Normalization and Pooling Validated Methods for Nextera and Nextera XT TruSeq Amplicon Cancer Panel And more! 26 Robotic NGS Sample Prep - Throughput estimates Procedure Samples / day Samples / day Single SPRI Cleanup 440 880 Double SPRI Cleanup 280 560 DNA Normalization 5000 10,000 Nextera PCR setup 1728 3456 Nextera DNA Library Prep 100 200