It’s Not Magic: Automated NGS Sample Preparation Zach Smith, MS Senior Application Scientist
Dec 18, 2015
It’s Not Magic: Automated NGS Sample Preparation
Zach Smith, MSSenior Application Scientist
NGS Workflows
Nucleic Acid Extraction and
Isolation
Next Generation Sequencing
Library Construction
Library Characterization
Nucleic Acid Extraction and Isolation: FormaPure and DNAdvance
Automated Nucleic Acid Characterization
Nucleic Acid Extraction and Isolation Wrap-Up
• Beckman Coulter automated liquid handlers combined with Beckman Coulter’s SPRI-based extraction chemistries allow for high-throughput DNA isolation in 96-well format
• Beckman Coulter automated liquid handlers allow for the quantification of DNA samples using fluorometric assays in 96 well format
Reduced vs Whole GenomesReduced Genome Whole Genome
Targeting Method Amplicon or Hybridization
Not Required
Advantages Lower cost, higher throughput, higher depth of coverage
Includes non-coding regions, more useful for assessing structural changes
Disadvantages Excludes non-coding regions, assay dependent on targeting method constraints
More expensive, lower depth of coverage, more computationally intensive
NGS Workflows
TagmentationNextera
A-tailingAdaptor Ligation
End Repair
TruSeq PCRFree
A-tailingAdaptor Ligation
PCR Enrichment
End Repair
TruSeq DNA/ TruSeq Nano
Illumina
Ion Torrent
Adaptor Ligation
PCR EnrichmentEnd RepairIon Torrent
NGS DNASeq Automation
Third Party Chemistry!New England Biolabs Illumina Kapa Biosystems
NGS Library Construction Automation
Automation vs. Manual: Not always the same
Common areas that might bring the most challenges:
– Labware Selection– Heating & Cooling– User Interventions– Tip Usage– Chemistry Specific
Labware Selection• Price vs. Automation compatibility
– Moving labware requires rigid plastic
• Shaking vs. Tip Mixing– Volume
• Cross contamination• Time
– Well shape• Round vs. V Bottom
• Tubes vs. 96-well plate• Dead Volume
Heating & Cooling• Peltier vs. Thermo cycler
– Peltier• Open Position: no sealing• Slow ramping/cooling speed• Less expensive• Not ideal for all applications (High temps)
– Thermo cycler• Expensive• No user intervention• Closes automatically: sealing on deck• Labware-few types/expensive
Tip Usage• Choosing Tip Type
– Limiting Tip Types (conserve waste)– Conductive and Non-Conductive
• Deciding when to re-use– Tip Re-Use
• MC and Span 8
Tip Mixing vs. Shaking• Shaking can:
– Reduce Time – Tip Usage– Provide uniform results
• Tip Mixing can:– Large Volume in well
• Cross contamination from splashing
– Labware or Well shape• V-Bottom PCR vs Round well
User Interventions• Natural Protocol Stop Points
– Safe Storage points– Pre/Post PCR break points
• Non-Protocol Stop Points– Allow for Off-deck incubations– Allow for recovery
Chemistry Specific Each kit or workflow can introduce a challenge.• How do you handle options for different
sample inputs/types?• How do you set up indexing?• How to pipette small volumes?
Protocol Options
NexteraXT Options• Number of
Samples• On Deck
Thermocycling• How much of the
method to run
Indexing• Requires Tracking
– Barcode readers, excel spreadsheets
• Prepare Indexed Plate Before-hand– Prepare indexed plate before-hand; stable at RT
during Library Construction– No evaporation issue of source– Plate barcodes can be utilized
Small Volumes• Knowing when to calibrate
– Accuracy vs. Precision• Dilute volumes
– 2 µL easier than 1 µL• Choosing Tip Type
– P50 and P20: Difference in tip type
P20: Shorter tip, higher volume with non-barrier
P50: Longer tip; easy access tubes
NGS Library Construction Wrap-Up
• Beckman Coulter offers excellent DNASeq library construction options in SPRIworks and SPRIworksHT platforms
• Beckman Coulter automated liquid handlers allow for the implementation of any number of available DNASeq library kits with easy, optimized workflows
NGS Library Characterization
NGS Library Characterization
96 qPCR quantified
NGS libraries
Single pool of 96 qPCR quantified
NGS libraries
NGS Library Characterization Wrap Up
• Beckman Coulter automated liquid handlers allow for the implementation of easy to use, integrated work-flows that allow for the purification of enriched NGS libraries with AmpureXP
• Accurate, high-throughput qPCR quantification of NGS libraries using a number of available kits
• Beckman Coulter automated liquid handlers make the pooling of large numbers of quantified NGS libraries prior to sequencing more efficient.
Summary: How Beckman Can Help
• Field Application Scientists: Knowledgeable Field Scientist can help you figure out the details
• Biomek Software: our software makes calibration easy, optimize individual workflows
• Biomek Hardware: Lots of configuration options, easy integration of devices
• Demonstrated Methods Available– Internal Development– Customer Collaborations
• Chemistry Partnerships
Questions?
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