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1 Click to edit Master title style DOPlify 1 PGS and PGD with DOPlify TM Web based Training UK Europe Monday 31 st July 2017 Introduction to RHS 2 RHS was founded more than 10 years ago in reproductive genomics and single cell analysis The company has developed and launched 2 products EmbryoCellect specifically for IVF embryo screening, enabled by DOPlify and DOPlify a platform technology spanning IVF, cancer and research applications and introduced PG Seq at ESHRE 2017 RHS has repositioned to broaden our focus, reflected in our recent name change from Reproductive Health Science to simply RHS The company is Adelaide based, with a specialised expert team of staff

2 Factors driving increased PGS use 3 Pre implantation Genetic Screening (PGS) screens embryos before transfer during IVF to determine whether they have the correct number of chromosomes. PGS increases the implantation rate and thus; Reduces the time to pregnancy Reduces the risk of miscarriage Supports single embryo transfer and avoids multiple pregnancies Allows the selection of healthy embryos for freezing for future use It is estimated that there are over 1 million embryos already in storage Overcomes the adverse impact of maternal age on IVF success Embryos derived from older mothers commonly fail to implant, PGS can attain similar implantation rates as young mothers Overview 4 Next Generation Sequencing (NGS) Whole Genome Amplification technology (WGA) DOPlify Data output PG Seq PGS whole chromosome aneuploidy Detection of segmental DNA aberrations mtdna quantification Combined PGD + PGS

3 NGS Workflow 5 DNA Fragmentation Add adapters Add index/barcode and amplify (5 8 cycles) Clean up, size selection Pool all samples Bioinformatics Sample DNA sequence Breadth and depth of coverage 6 DNA sequence DNA sequence Breadth = the proportion of bases in the target sequence that are represented Depth = the number of times each base is represented

4 NGS Terminology Read Length 7 Range from 36, 50, 75, bp Single read or paired end reads (eg 1x75 or 2x75) Single Paired end The shorter the read length, the faster the run time and the less detailed the information Longer reads provide more information and are more suited to SNP or targeted gene sequencing NGS Terminology Capacity 8

5 NGS Terminology Reads per sample 9 Each run generates a finite amount of data The more samples run, the less data per sample. Total DNA data Sample number Sample number Factors impacting coverage Read length 10 Genome coverage is greater using longer read lengths, allowing increased sample multiplexing. Modelling demonstrates that by increasing the read length from 36bp to 75bp, greater genomic coverage is achieved even when the number of samples in the run is doubled from 24 to 48. % Unmapped Data loss is greater using short read lengths Read Length Coverage of Human Genome Genome Coverage 1x36bp 1x75bp Number of samples per run RHS, ESHRE, 2017

6 The need for Whole Genome Amplification 11 Single cell 5 cells Microarray Standard NGS Low template NGS ,000 10, ,000 Cell equivalents of DNA input required (log scale) Commercially available WGA kits 12 PCR based WGA kits SurePlex, PicoPlex, GenomePlex Ampli1 DOPlify Isothermal WGA kits REPLI g TruePrime Hybrid WGA kits MALBAC LIANTI Polymerase Characteristics Performance in optimal buffer & temperature Processivity Fidelity (error rate) 3 5 Proof reading ability

7 WGA Kit Protocol Steps & Times 13 Telenius DOP DOPlify MALBAC REPLI g Ampli1 TruePrime PicoPlex GenomePlex SurePlex Protocol Time (Minutes) Hands on Incubation Differences in WGA DNA products 14 M NC 1kb 0.5kb DOPlify 200bp 3kb 60 80ng/ul SurePlex 200bp 1kb 25 65ng/ul MALBAC 300bp 2kb 30 55ng/ul REPLI g 2 100kb Up to 800ng/ul Longer fragments may copy entire genes, good for linkage analysis due to high number of SNPs per copy Shorter fragments are better for detecting copy number changes Li et al, 2015

8 DOPlify mitochondrial DNA genome 15 PicoPlex 1 PicoPlex 2 DOPlify 1 DOPlify 2 Total mapped reads to human genome (hg19) 16,200,000 9,800,000 6,500,000 8,200,000 mtdna Coverage (read length x reads/genome size) x 101 x 29 x 3348 x 3702 mtdna Percent coverage at x1 92 % 67% 100 % 100 % Percent of total mapped reads DOPlify PicoPlex DOPlify amplification similar to bulk DNA 16

9 Factors compromising WGA 17 Sample collection procedure/biopsy Damage to the cell or DNA Cell cycle phase Sample storage & transport DNA integrity/degradation Transfer buffers or PCR inhibitors Incomplete cell lysis and DNA template release WGA bias or nucleotide incorporation errors Cell cycle DNA copy number changes 18 DNA copy number is recorded as gains and losses, and during active DNA replication the variability in greater in comparison to other phases of the cell cycle. Non replicating DNA G1 and G2/M phase Replicating DNA S phase = more copies than reference = same as reference, 2 copies = less copies than reference Van Der Aa et al, 2013

10 R&D Conference abstracts Combined PGD and PGS: Enrichment of PGD genes during whole genome amplification (PGDIS 2016) 2. Combined PGD/PGS Using Whole Genome Sequencing in a Model of Blastomere and Trophectoderm Biopsy (PGDIS 2016) 3. Whole Genome Amplification:No Method is the Same (FSA 2016) 4. Validation of EmbryoCellect with SurePlex amplified embryo biopsies (PGDIS 2017) 5. Combined PGS and PGD for Thalassemia (PGDIS 2017) 6. Combined PGD and PGS by NGS on the same biopsy using a single index (PGDIS 2017) 7. Mitochondrial genome coverage for copy number determination and detection of disease; the impact of WGA (PGDIS 2017) 8. Development of a 5 hour PGS protocol for a day 5 fresh transfer (PGDIS 2017) 9. Validation of a high throughput, low cost NGS PGS assay: impact of library preparation and read length on resolution (ESHRE 2017) 10. WGA and NGS read length significantly impact mitochondrial characterisation (ASRM 2017) DOPlify Click to edit WGAMaster title style 20 Cell Lysis Whole Genome Amplification DOP PCR QC step Agarose Gel Electrophoresis

11 DOPlify Kit Contents 21 Kit Codes Component Cap colour RHS reactions PCR-grade H 2O White Cell Lysis Enzyme Cell Lysis Buffer WGA Polymerase WGA PCR Buffer Primer Yellow Yellow Red Red Red Storefrozenat-20 C Input Specifications 22 Cell Types Single blastomeres polar bodies trophectoderm cells Amniocytes lymphocytes cultured cells DNA (30pg/µl) Number of Cells Single cells or multi cell aliquots Cell Collection Method Transfer buffer volume (<2 µl) Compatible Buffers Recommended cell transfer buffers 10 mm Tris HCl (ph 8.0) (No EDTA) and PBS (Mg 2+, Ca 2+ free and BSA free)

12 DOPlify WGA workflow 23 Sample collection o Place the sample into a PCR tube in <2 μl of buffer o Mark the sample location on the tube Lysis o Add 3 μl of lysis solution above the sample o Tap the PCR tube to allow the lysis solution to roll over the sample o Incubate for 15 min according to the lysis program Whole Genome Amplification (WGA) o Add 22 μl of PCR master mix to the lysed sample o PCR for 2.5 hours using the WGA PCR program o Assess WGA by agarose gel electrophoresis Cell Lysis WGA Protocol Time (minutes) Hands on Incubation Cell lysis 24 Protocols: RHS DOP-PCR Whole Genome Amplification Cell Lysis Estimated Time: 4 samples + 1 No Template Control (NTC) Hands on: 10 minutes Thermocycler: 15 minutes Total duration: 25 minutes Component Volume for 1 lysis reaction Recommended minimum volume (6.7x master mix) Cap colour PCR grade H 2 O 2.7 µl 18 µl White Cell Lysis Buffer 0.15 µl 1.0 µl Yellow Cell Lysis Enzyme Dilution µl 1.0 µl Total volume 3.0µl 20.0 µl Process Temperature Duration Cycles Lysis 75 C 10 min Heat 95 C 5 min inactivation 1 4 C Hold

13 Whole Genome Amplification 25 Component Volume for 1 Cap colour WGA reaction PCR-grade H 2 O 6.5 μl White WGA PCR Buffer 12.5 μl Red Primer 2.5 μl Red WGA Polymerase 0.5 μl Red Total volume 22 μl Step Temperature Duration Cycles Initial denaturation 95 C 5 min 1 Denaturation 98 C 20 sec Annealing 25 C 1 min 30 sec 8 Ramp to 72 C 1 C/4 sec Extension 72 C 1 min Denaturation 98 C 20 sec Annealing 58 C 1 min 21 Extension 72 C 1 min Final extension 72 C 1 min Cooling 15 C Hold 1 WGA Quality Control 26 A failed WGA amplification is indicated by the presence of primer dimers, but no evidence of the larger amplification products (see WGA Example 2, lane 4). Possible causes are that the sample was not successfully transferred to the PCR tube or that the sample was located in the PCR tube above the lysis and PCR reagents. Failed samples should be discarded. Poor WGA amplification is indicated by smears with lower intensity or with PCR products that are notably larger or smaller than the expected size range observed for the other samples on the same agarose gel (see WGA Example 2, lane 6). The results from these samples should be interpreted with caution and it is recommended that these samples are discarded WGA Example 1: Lane 1: DNA Ladder (100bp) Lanes 2-5: Amplified single cells Lanes 6-9: Amplified 30 pg genomic DNA reference Lane 10: PCR no template control (NTC) Lane 11: DNA Ladder (100bp) WGA Example 2: Lanes 1, 2, 3, 5, 7, 8: Amplified single cells Lane 4: Failed WGA reaction Lane 6: Poor WGA reaction Lane 9: NTC Lane 10: DNA Ladder (100bp)

14 Laboratory Set up for PGS Potential for PCR contamination 28 Non-discriminate amplification of DNA Target = Biopsy sample CONTAMINATION is highly possible Source: Embryologist, laboratory technician Shared equipment other WGA DNA samples

15 Single Cell laboratory set up 29 To have the greatest confidence in the results and reduce the risk of DNA contamination of biopsy samples, there must be complete control of equipment, NO SHARING CLEAN LAB NO DNA CONTAMINATION Highly recommended set up: Pre PCR Area Dedicated pipette set only for cell lysis and PCR master mix set up Physically isolated single cell laboratory room Dedicated PCR thermocycler Post PCR Area Amplified DNA is processed/handled eg gel electrophoresis, library preparation, DNA quantification, NGS PG Seq Click to edit Workflow Master title style Cell Lysis Whole Genome Amplification DOP PCR QC step Agarose Gel Electrophoresis Library Preparation Sequencing Run Data Analysis 30

16 PG Seq for PGS by NGS 31 47,XY,+9 48,XXY,+21 PG Seq TM Validation Data interim data set Total Correct FP FN FP + FN Single cell % (97) 5% (5) 0% (0) 1% (1) 5 cell % (93) 0% (0) 1% (1) 0% (0) PG Seq TM Validation Data due for release ASRM 2017 Limitations of single cell PCR 32 There are 2 main issues that affect PCR performance: Allele Drop Out (ADO) ie where an allele fails to amplify = false negatives for detection Bias (over & under representation) = false negatives and positives for quantification This is an issue for single gene PCRs eg PGD but also occurs in whole genome amplification 19

17 PG Seq CNV detection accuracy 33 An independent benchmarking study undertaken by the University of Ghent demonstrated that only PicoSeq and DOPlify were able to accurately detect CNVs in 1, 3 or 5 cell samples from the Loucy cell line (Deleye et at, 2017) Specificity (%) PPV (%) PicoSeq DOPlify Ampli ± ± 13.7 RepliG 96.3 ± ± 10.1 DOPlify PicoSeq Segmental DNA aberrations X Y Chromosome GM09552 cell line chromosome 8 7Mb loss and 31Mb gain.

18 BRCA1 2bp deletion 35 Single Cell 5 Cell gdna Also SNPs for phasing in the absence of sequence coverage DOPlify mtdna quantification 36 5 cell cell lines (Coriell) Embryo viability TE Biopsies mtdna reads/total reads Euploid N=24 Aneuploid N=65 RHS, unpublished Ravichandran et al, 2017

19 Combined Click to edit PGD Master + PGStitle style 37 Why combine PGD with PGS? 38 PGD and PGS are not routinely combined PGD unaffected embryos can be aneuploid Only 26% of PGD unaffected embryos were euploid (Goldman et al, 2016) So what are the options to combine PGD and PGS?

20 Strategies for PGD + PGS 39 Direct PCR WGA for PGS WGA +PGD No PGS The region of interest may be amplified during WGA ADO rate higher than PGD 1 PCR; 2 results ADO rate equivalent to PGD DOPlify TSE patented approach DOPlify Lysis 3. WGA + Gene specific PCR MiSeq 2x75bp kit 40 sample multiplex Economical High throughput Universal WGA 2. DOPlify WGA This approach achieves: Accurate WGA for PGS 100% target coverage with >200x read depth for accurate PGD It is compatible with a range of PGD methods including PCR, electrophoresis, Sanger sequencing, NGS

21 NGS sample preparation for MiSeq 41 WGA WGA + TSE WGA + TSE + Amplicon 1:10 WGA + TSE + Amplicon 1:20 PCR Amplicon Comparison of 3 strategies sample multiplex MiSeq 2x75bp reads DNA sequence WGA only < 1x depth of coverage Exon 3 Exon 2 Exon 1 thalassemia

22 Comparison of 3 strategies sample multiplex MiSeq 2x75bp reads DNA sequence WGA only < 1x depth of coverage WGA + TSE 5x Exon 3 Exon 2 Exon 1 thalassemia Comparison of 3 strategies sample multiplex MiSeq 2x75bp reads DNA sequence WGA only < 1x depth of coverage WGA + TSE 5x WGA + TSE & amplicon pool 200x Exon 3 Exon 2 Exon 1 thalassemia

23 Breadth of coverage 100% 45 DNA sequence Percent of PCR Product (%) Exon 1&2 Exon 3 Exon 1&2 Exon 3 Exon 1&2 Exon 3 WGA only (n=6) HBB WGA + TSE (n=7) WGA + TSE + PCR product 1:20 (n=5) Percent of PCR product (%) WGA only (n=8) WGA+TSE (n=6) HLA A WGA+TSE+PCR product 1:20 or 1:10 (n=4) (n=5) PCR product (n=2) 40 sample multiplex MiSeq 2x75bp reads SNP analysis 46 HLA typing WGA + TSE + Amplicon 1:20 Exon 2 Exon 3 Frequency of A allele is 63.37

24 Competitive advantages of RHS products 47 DOPlify Kit DOPlify Kit for combined PGD + PGS PG Seq Workflow Simple protocol with only 2 sample tube openings Minimises risk of contamination Reduces hands on time Less steps than other WGA kits Clear QC prior to expensive downstream analysis Fast protocol that matches the fastest on the market Latest generation reagents that are accurate and produce high yields of the DNA template Robust kit shippable on ice packs and with a long shelf life Scalable and economical solution for PGS Flexible WGA system that uniquely allows the addition of primers to copy specific regions of clinical significance concurrently rather than sequentially Disease causing genetic mutations, HLA markers, STR markers (patent filed) Superior coverage demonstrated by human mitochondrial genome data RHS Unique Genomic Solutions 48 RHS Team Matthew Brockman Christine Robinson Sandra Protopsaltis Kimberly Warren Sequencing Collaborators University of NSW (Australia) The Ramaciotti Centre (Australia) SAHMRI (Australia) ACRF (Australia) EmbryoCellect, PG Seq and DOPlify are Research Use Only product and are not to be used for diagnostic procedures For more information on the products use, limitations, and licenses: info@rhsc.com.au