Large scale plant genomics: breaking the sample preparation barrier

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