Improved Chemistry for NGS Library Cleanup and Size Selection Speakers: Charles Cowles, PhD & Curtis Knox Promega Corporation
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
Size-Selective Purification Methods and Usage Promega Corporation
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
Sample Cleanup Single-sided size-selection Remove fragments below a chosen size Useful for PCR cleanup, primer/adapter removal, buffer exchanges Promega Corporation 5
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) 100 150 250 350 475 550 650 800 1,000 ProNex Size-Selective Purification System ratios shown Promega Corporation 6
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
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 275 350 450 550 600 Size (bp) ProNex Size-Selective Purification System ratios shown Promega Corporation 8
Possible Areas for Library Prep Improvement Promega Corporation
Loss of DNA During Multiple Purification Steps % of Initial Input 100 90 80 70 60 50 40 30 20 10 0 1 2 3 4 5 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
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
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
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
Developing a Better Size-Selection Chemistry Promega Corporation
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
Efficiency and Reproducibility Comparison 120 100 0.9% CV % Recovery 80 60 6.0% CV 1µg input, 100bp ladder 3x ratio ProNex chemistry added 40 20 1.8x ratio AMPure XP beads added 0 ProNex Chemistry AMPure XP Promega Corporation 16
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
Magnetic Response Time and Viscosity Promega Corporation
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% 0 100 200 300 400 500 600 Time (s) Promega Corporation 19
Comparative Magnetic Response Time Promega Corporation 20
Comparative Viscosity and Ease of Pipetting Promega Corporation 21
Comparative Library Prep and Sequencing Experiments Promega Corporation
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
Final Library Yield Total Yield (ng) Total Yield (ng) Promega Corporation 24
Final Library Size Distributions Promega Corporation 25
Final Library Size Distributions Promega Corporation 26
Final Library Sequencing Fragment Size Distribution Promega Corporation 1
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 234 239 70 465 AMPure XP 1000ng 318 358 167 1252 ProNex System 100ng 224 229 64 524 AMPure XP 100ng 305 348 164 1238 ProNex System 10ng 222 227 63 553 AMPure XP 10ng 299 345 166 1271 ProNex System 1ng 221 226 65 605 AMPure XP 1ng 279 324 158 1240 Promega Corporation 28
User-Generated Library Data Promega Corporation
User Data Multiple Cleanup Steps in Library Prep FFPE Sample Promega Corporation 30 AMPure XP Recovery (ng) ProNex System Recovery (ng) Difference (ng) Difference (%) 1 380 524 +144 +38% 2 369 656 +287 +78% 3 464 564 +100 +22% 4 224 416 +192 +85% 5 720 704-16 -2% 6 684 812 +128 +19% 7 340 332-8 -2% 8 165 310 +145 +88% 8 separate formalin-fixed paraffinembedded (FFPE) samples with varying DNA input levels 3 cleanups each sample throughout process Average 40% increase in yield
User Data Replicate Study DNA yield post clean-up (n=16) 600 500 400 300 200 100 0 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
User Sequencing Data Ion Torrent Amplicon Library 120% % Library DNA Retention 300 Average Library Concentration (pm) 100% 250 80% 200 60% 150 40% 100 20% 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
User Sequencing Data Ion Torrent Amplicon Library Pre-cleanup Post-cleanup ProNex System Pre-cleanup Post-cleanup AMPure XP Promega Corporation 33
Applications of ProNex Size-Selective Purification System Promega Corporation
High Molecular Weight Purification Promega Corporation
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
Circulating Cell-Free DNA (ccfdna) Cleanup Promega Corporation
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 25 100 250 400 500 1000 2500 5000 10000 25 100 250 400 500 1000 2500 5000 10000 Promega Corporation 38
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
Thank You! For questions, please contact Promega Technical Services: techserv@promega.com Promega Corporation 40