1. It’s Not Magic: Automated NGS Sample Preparation Zach Smith, MS Senior Application Scientist

2. NGS Workflows Nucleic Acid Extraction and Isolation Next Generation Sequencing Library Construction Library Characterization

3. Nucleic Acid Extraction and Isolation: FormaPure and DNAdvance

4. Automated Nucleic Acid Characterization

5. 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

6. Reduced vs Whole Genomes Reduced GenomeWhole Genome Targeting MethodAmplicon or Hybridization Not Required AdvantagesLower cost, higher throughput, higher depth of coverage Includes non-coding regions, more useful for assessing structural changes DisadvantagesExcludes non- coding regions, assay dependent on targeting method constraints More expensive, lower depth of coverage, more computationally intensive

7. NGS Workflows Tagmentation Nextera A-tailing Adaptor Ligation End Repair TruSeq PCRFree A-tailing Adaptor Ligation PCR Enrichment End Repair TruSeq DNA/ TruSeq Nano Illumina Ion Torrent Adaptor Ligation PCR Enrichment End Repair Ion Torrent

8. NGS DNASeq Automation Third Party Chemistry! New England Biolabs Illumina Kapa Biosystems

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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?

16. Protocol Options NexteraXT Options Number of Samples On Deck Thermocycling How much of the method to run

17. 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

18. 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

19. 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

20. NGS Library Characterization

21. 96 qPCR quantified NGS libraries Single pool of 96 qPCR quantified NGS libraries

22. 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.

23. 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

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