Accurate Nucleic Acid Analysis is Critical to Successful Sequencing. Steve Siembieda VP Commercialization November 10, 2016

Transcription

1 Accurate Nucleic Acid Analysis is Critical to Successful Sequencing Steve Siembieda VP Commercialization November 10,

2 Advanced Analytical Privately-held instrumentation company, established in 1998 Sales/Technical/Manufacturing office in Ankeny, IA Sales/Technical office in Germany and France Instruments in operation worldwide

3 AATI Partners

4 World Leaders in Parallel CE

5 Fragment Analyzer Now the Industry Standard Developed to address key bottlenecks that slow results generation Parallel Capillary Electrophoresis platform Unique design features to improve workflow and changed how QC is done

6 Overall Operations

7 Unique Design Features Widest range of kits/applications/uses Switch between 2 different gels matrices Hold 3 x 96-well sample plates 12, 48 or 96 capillary array and lengths Flexible and dependable operations High quality analytical software

8 Reagent Kits

9 Reagent Kits

10 Reagent Kits

11 New Kits in 2016 DNF-464 Large Fragment 50Kb Analysis Kit DNF-467 Genomic DNA 50Kb Analysis Kit DNF-477 Small Fragment Analysis Kit DNF-470 Small RNA Analysis Kit CRISPR Kits For Agarose gels and Fragment Analyzer

12 Large Fragment 50Kb Kit DNF-464 Designed for better sizing of large fragment smears through 50,000bp Dynamic range is 50pg/µL - 5ng/µL Normalization is required to accurately assess small differences between large fragment smears

13 Large Fragment 50Kb Kit New Ladder with fragments to 48,500bp Upper marker at 200,000bp equivalent Better sizing and quantification through range

14 Smear Analysis Settings - The smear analysis feature shows average sizing over the defined range - The average size can be put into the PacBio Binding Calculator

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16 Fragment Analyzer Benefits Accurately assess the size, quality and quantity of large fragment smears to 50Kb Replace overnight PFGE separations Prepare a library a faster

17 Incoming Raw Material Analysis Genomic DNA FFPE DNA cfdna Total RNA Ribodepleted RNA Messenger RNA FFPE RNA Small & microrna

18 Genomic DNA 50Kb Kit DNF Designed for more accurate analysis of genomic DNA smears Input DNA range - 25ng/µL - 250ng/µL High input concentrations Uses single, large 200 fold dilution 1 µl sample instead of 2 µl

19 Qubit Concentration, ng/µl Genomic DNA Analysis Reproducibility of gdna quantitation y = x R² = Fragment Analyzer Concentration, ng/µl High Molecular Weight DNA Coriell Biorepository sample Linear relationship between Fragment Analyzer as compared to the Qubit. The quantitation accuracy is ±15% and precision ±20%.

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21 Small RNA Analysis Kit DNF Newly developed kit for analyzing small RNAs including micrornas Sizing range is 5nt-200nt Analysis dynamic range is 50pg/µL to 2,500pg/µL Separation time is 18 minutes

22 Human Liver Small RNA Value [Small RNA], pg/ul [microrna], pg/ul 76.1 % microrna 10.2 trna size, nt 66/80 5S size, nt S size, nt 160 Expected Concentration 1 ng/µl

23 Synthetic Guide RNA QC Guide quality assessment Improper handling or contamination Improve sensitivity over agarose gels Fresh sgrna Heated at 90C for 30 minutes

24 Small Fragment Analysis Kit DNF Designed for small fragments and small fragment smears analysis High Sensitivity Kit Sizing range 50bp 1,500bp Fast run times

25 Small Fragment Analysis Kit Small Fragment Ladder

26 Sheared DNA Quantification 1ng/µL smear average size 450bp

27 Small Fragment Analysis Kit Small RNA NGS library preparation

28 cfdna Separation

29 cfdna Application Note

30 CRISPR Hot area of study Typical Goals Modify a specific gene or genes Mitigate off-target effects Typical Outcomes Identify cells with desired changes Isolate/propagate individual edited cells

31 Fragment Analyzer for CRISPR Greater sensitivity allows for lower DNA inputs requirements Automated software tools for calculating percent cleavage percent mutated of pooled samples zygosity of individual cell lines No need for costly probes

32 CRISPR Screening Kits CRISPR Gel Kit DNF CP Mutation Discovery Gel dsdna reagent kit Analysis range bp AccuCleave T7 CE Kit For automated digestion of heteroduplex fragments Optimization completed Control Amplification Kits (C2 & C15) 2 fragment control & 15 fragment control 32

33 Agarose Kits

34 AccuCleave T7 Kit Examples Control Fragment mixtures at different molar concentration ratio. Lanes 2 and 3, 95/5 mix of WT/-2bp Lanes 4 and 5, 50/50 mix of WT/-2bp Lanes 6 and 7, 50/25/25 mix of WT/-2bp/+2bp

35 Fragment Analyzer Leads The first instrument that can Holds THREE 96 well plate Holds TWO gels Seamlessly switch between different applications Vary the separation length and number of samples per run Only instrument that has kits for quantification and kits for qualification (CRISPR) 35

36 Built on the Fragment Analyzer frame Holds a 12 capillary array Femtogram level sensitivity Separation of DNA through 200,000bp FEMTO Pulse

37 HMW DNA Separation Separation time ~1 hour Average bp

38 Femtogram Sensitivity Detect to 5 fg/µl fragments without PCR end labeling 40fg/µL = 120,000 molecules 20fg/µL = 60,000 molecules 10fg/µL = 30,000 molecules 5fg/µL = 15,000 molecules 300 bp fragment

39 Fragment Analyzer FOR TODAY The current Industry Standard Use for library preparations and incoming raw materials evaluation Speed up library preparation FEMTO Pulse The Next Standard Available WW Q x more sensitivity Separate and size HMW genomic DNA, BACs & fragments

40 FEMTO Pulse Fragment Analyzer Industry standard Replace 2 overnight PFGE separation Speed up library preparation Other uses for evaluating smaller libraries and raw materials Endorsed COMING SOON The Next Industry Standard Available WW 2017 Up to 100x more sensitive Separate and size HMW genomic DNA, BACs & smears

41 Thank You! Need more information? Steve Siembieda, VP Commercialization