Improved Chemistry for NGS Library Cleanup and Size Selection Speakers: Charles Cowles, PhD & Curtis Knox

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1 Improved Chemistry for NGS Library Cleanup and Size Selection Speakers: Charles Cowles, PhD & Curtis Knox Promega Corporation

2 Agenda What is size-selective purification and how is it used? Why is there a need for better library sample cleanup and size selection? Goals for improvement Comparative data Applications in high molecular weight cleanup and circulating cell-free DNA (ccfdna) applications Promega Corporation 2

3 Size-Selective Purification Methods and Usage Promega Corporation

4 Why is Size-Selective Purification Used? NGS library preparations require not only the ability to clean the samples, but selectively remove only certain size DNA fragments Buffer exchanges or sample cleanups to avoid downstream interference Removal of primers, adapters or other small fragments Fragments that are too small/large can interfere with proper sequencing on NGS instruments Accurate size selection is key to optimal NGS sequencing results. Promega Corporation 4

5 Sample Cleanup Single-sided size-selection Remove fragments below a chosen size Useful for PCR cleanup, primer/adapter removal, buffer exchanges Promega Corporation 5

6 Single-Sided Cleanup Example Ratios ProNex Chemistry Ratio(v/v) Input 3x 2x 1.5x 1.3x 1.2x 1.15x 1.1x 1.05x 1x Approximate Size Cutoff (bp) ,000 ProNex Size-Selective Purification System ratios shown Promega Corporation 6

7 Dual Size-Selection Useful for removal of DNA above and below a chosen size range Used in whole genome sequencing or amplicon libraries when undesired fragments or off-target amplicons need to be removed Promega Corporation 7

8 Dual Side-Selection Example Ratios First/Second 1.2/0.4x Ratio 1.1/0.35x Ratio 1.05/0.35x Ratio 1.0/0.3x Ratio 0.95/0.3x Ratio Average Library Size (bp) ProNex Size-Selective Purification System ratios shown Promega Corporation 8

9 Possible Areas for Library Prep Improvement Promega Corporation

10 Loss of DNA During Multiple Purification Steps % of Initial Input Purification Round Current methods = 20-30% loss with each purification step Many library prep methods incorporate serial purifications Loss of DNA requires greater starting material or more PCR cycles Promega Corporation 10

11 High Viscosity of Chemistry = Pipetting Error Chemistry sticks to tips Retention is variable, resulting in lower reproducibility Requires special liquid handling classes and programming in automated protocols Promega Corporation 11

12 Speed of Magnetic Response Current chemistry resin is slow to respond to magnet Requires greater incubation periods Can leave resin in suspension at time of supernatant removal 2 minutes elapsed time on magnet Promega Corporation 12

13 Specificity of Size Selection Current methods struggle with base-lining the large fragments Large Fragments to be Removed Long fragments can interfere with proper clustering Critical for newer Illumina platforms (NextSeq, NovaSeq) that do not tolerate large fragments on patterned flow cells Whole genome library, generated with NEBNext Ultra II DNA Library Prep Kit for Illumina, centering at 325bp per kit manufacture protocol. Size-selection performed with standard AMPure XP beads. Promega Corporation 13

14 Developing a Better Size-Selection Chemistry Promega Corporation

15 Goals for Improving Size-Selection Chemistry Greater % recovery of input DNA More specific size selection Lower carryover of HMW DNA in dual size-selection methods Lower viscosity, resulting in greater ease of use, better reproducibility, and compatibility with automation platforms Inclusive kit: contains wash buffer and elution buffer Promega Corporation 15

16 Efficiency and Reproducibility Comparison % CV % Recovery % CV 1µg input, 100bp ladder 3x ratio ProNex chemistry added x ratio AMPure XP beads added 0 ProNex Chemistry AMPure XP Promega Corporation 16

17 Serial Purification Comparison Sample cleanup, n=8 replicates per treatment ProNex System = 3x ratio, AMPure XP beads = 1.8x ratio 200bp ladder used, all fragments expected to be retained Promega Corporation 17

18 Magnetic Response Time and Viscosity Promega Corporation

19 Comparison of ProNex and AMPure XP Magnetic Bead Response Rates 100% 99% 98% % of Beads Sequestered 97% 96% 95% 94% 93% ProNex PromegaSystem AMPure AMPureXPXP 92% 91% 90% Time (s) Promega Corporation 19

20 Comparative Magnetic Response Time Promega Corporation 20

21 Comparative Viscosity and Ease of Pipetting Promega Corporation 21

22 Comparative Library Prep and Sequencing Experiments Promega Corporation

23 Comparative Library Prep Study E. coli gdna input Library prep with NEBNext Ultra II DNA Library Prep Kit for Illumina Libraries centered at 325bp (including adapters) 1000ng, 100ng, 10ng and 1ng input levels (3 replicates of each) Size selection performed with ProNex System or AMPure XP Sequencing performed on an Illumina MiSeq Goals: Compare library yield, shape and final sequencing metrics Promega Corporation 23

24 Final Library Yield Total Yield (ng) Total Yield (ng) Promega Corporation 24

25 Final Library Size Distributions Promega Corporation 25

26 Final Library Size Distributions Promega Corporation 26

27 Final Library Sequencing Fragment Size Distribution Promega Corporation 1

28 Final Sequencing Metrics Median Insert Size (bp) Mean Insert Size (bp) Insert Size Standard Deviation (bp) Width of 99 % of Insert Sizes (bp) ProNex System 1000ng AMPure XP 1000ng ProNex System 100ng AMPure XP 100ng ProNex System 10ng AMPure XP 10ng ProNex System 1ng AMPure XP 1ng Promega Corporation 28

29 User-Generated Library Data Promega Corporation

30 User Data Multiple Cleanup Steps in Library Prep FFPE Sample Promega Corporation 30 AMPure XP Recovery (ng) ProNex System Recovery (ng) Difference (ng) Difference (%) % % % % % % % % 8 separate formalin-fixed paraffinembedded (FFPE) samples with varying DNA input levels 3 cleanups each sample throughout process Average 40% increase in yield

31 User Data Replicate Study DNA yield post clean-up (n=16) FFPE DNA Library Clean-up ProNex System 2x ratio AMPure XP 1x ratio 16 replicates for each cleanup method Single cleanup for each Total DNA yield measured by QuantiFluor dye 4x reduction in standard deviation Promega Corporation 31

32 User Sequencing Data Ion Torrent Amplicon Library 120% % Library DNA Retention 300 Average Library Concentration (pm) 100% % % % % 50 0% 0 ProNex ProNex System AMPure XP XP ProNex ProNex System AMPure XP XP Serial sample cleanup method: ProNex System: 3x cleanup, followed by 2.75x size selection AMPure XP beads: 1.5x cleanup, followed by 1.3x size selection Promega Corporation 32

33 User Sequencing Data Ion Torrent Amplicon Library Pre-cleanup Post-cleanup ProNex System Pre-cleanup Post-cleanup AMPure XP Promega Corporation 33

34 Applications of ProNex Size-Selective Purification System Promega Corporation

35 High Molecular Weight Purification Promega Corporation

36 ProNex System = More DNA, Greater Selectivity Less carryover 1,000bp peak ProNex AMPure XP Better recovery ProNex chemistry shows less carryover of DNA species that are 600bp and less, indicating more selectivity ProNex chemistry yields more DNA of 1000bp (target cutoff) and higher Ratios used for non-peg containing reactions will be higher than shown here Promega Corporation 36

37 Circulating Cell-Free DNA (ccfdna) Cleanup Promega Corporation

38 Cleaner ccfdna by Size Selection Use of size selection to eliminate larger material decreases background without affecting levels of the 170bp fragment ProNex System used to exclude materials greater than 400bp Size Marker Plasma Size Marker 170bp Serum Post Size Marker Post Size Marker 170bp Promega Corporation 38

39 Summary Development of a new chemistry for NGS library cleanup and size selection provides important improvements in performance: Significantly increased final library yields Substantially reduced solution viscosity compared to standard products, resulting in: faster magnetic response time easier pipetting more reproducible results compatibility with automation platforms Enhanced dual-size selection, with more complete removal of high molecular weight DNA from final library Better Reproducibility Higher Recovery % Better NGS Libraries More Accurate Size Selection Promega Corporation 39

40 Thank You! For questions, please contact Promega Technical Services: Promega Corporation 40