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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