ATCCG GGTC. Whole Genome Amplification DOPlify TM Launch

Transcription

1 ATCCG GGTC Whole Genome Amplification DOPlify TM Launch Melinda Jasper, PhD CSO 23 rd August 2016

2 Forward Looking Statements 2 Any forward looking statements in this presentation have been prepared on the basis of a number of assumptions which may prove incorrect and the current intentions, plans, expectations and beliefs about future events are subject to risk, uncertainties and other factors, many of which are outside Reproductive Health Science Limited s control. Important factors that could cause actual results to differ materially from any assumptions or expectations expressed or implied in this presentation include known and unknown risks. As actual results may differ materially to any assumptions made in this presentation, you are urged to view any forward looking statements in this presentation with caution. This presentation should not be relied on as a recommendation or forecast by Reproductive Health Science Limited, and should not be construed as either an offer to sell or a solicitation of an offer to buy or sell shares in any jurisdiction.

3 Welcome to Reproductive Health Science Ltd. Since its inception, RHS has maintained a consistent focus on improving health and research outcomes and has built an experienced team and significant know-how in the development of whole genome amplification and single cell technology tools. It is this expertise that has gone into RHS DOPlify kit. RHS is located in the Bio Innovation SA Incubator in the heart of the Thebarton biotechnology precinct in Adelaide, South Australia. Dr Michelle Fraser, CEO and Managing Director Reproductive Health Science Ltd

4 Population Diversity 4

5 Understanding cell heterogeneity 5 Complex Multicellular Organism Specialized organs Single cell

6 Modern IVF and collection of biopsy 6 Day 3 Day 5 Biopsy & PGS Egg and sperm samples collected and egg fertilized Embryo selected for transfer, remaining embryos can be frozen or matured further PGS increases pregnancy rates by enabling the selection of chromosomally normal embryos Fiorentino, 2012 Biopsy & PGS (Last opportunity) Embryo selected for transfer, remaining embryos frozen for future use

7 Stem cells 7 Stem cell transplantation Limited template In vivo-post-transplantation monitoring in eg hematopoietic stem cell transplantation (HSCT) To predict negative events, such as disease relapse, graft rejection and graft-versus-host disease, in order to intervene with appropriate therapy. In vitro monitoring

8 Single cell analysis in cancer 8 Genetic Diversity within a tumour Growth characteristics Prediction of treatment response Metastatic potential Detection and characterisation of circulating tumour cells Identification and prognosis of primary source Mutation rate compared to tumour Prediction and monitoring of treatment response Treatment effectiveness and recurrence Permission obtained from Thieme Schilling, D. et al. Mechanisms of tumour cell dissemination and methods for detection of circulating tumour cells in transitional cell carcinoma. Aktuelle Urol. 42, (2011). Schilling, D. et al. (2012) Isolated, disseminated and circulating tumour cells in prostate cancer Nat. Rev. Urol. doi: /nrurol

9 Forensic applications 9 Commonly used STR markers require 250pg of starting DNA template Termed low copy number analysis if less than 100pg of DNA available More technical artefacts, evidence not widely accepted in court By using the RHS combined whole genome amplification and single gene enrichment protocol, it may be possible to obtain genetic fingerprints using STR markers from limited template Identification of individuals can be achieved using mitochondrial DNA, which is maternally inherited

10 Research applications 10 Environmental DNA and conservation Human microbiome Using whole genome amplification, all of the DNA in a sample will be amplified, providing a more abundant template for analysis. The microbiome of individuals may differ due to such factors as diet, environmental influences and genetics. The effects of therapeutic treatments, stress and the ability to restore the microbiome rely on the accurate characterisation of its contents

11 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)

12 Technical challenges 12 Acquiring high-quality data has 4 technical challenges: 1 Efficient physical isolation of the cells 2 Amplification of DNA sufficient quantities 3 Cost-effective downstream application 4 Interpreting the data within the context of bias and errors introduced during the above steps

13 Amplification of DNA - limitations 13 2 main issues: Allele Drop Out (ADO) i.e. where an allele fails to amplify = false negatives for detection Bias (over- & under-representation) = false negatives and positives for quantification RHS has undertaken focussed Research & Development to optimise DOPlify TM, including consideration of; DNA coverage Ease of use Shipping & storage stability

14 DOP-PCR WGA 14 Primer -anchor at the 3 end, 6bp degenerate sequence in the middle (i.e. a mixture of As, Cs, Ts and Gs in random order), another anchor at the 5 end. PCR program -small number of low stringency cycles, where the primer binding temperature is low, allowing the 3 anchor to bind indiscriminately across the entire genome while the rest of the DOP primer does not need to bind (PCR step 1). High stringency cycles where the entire primer sequence is used (PCR step 2).

15 R&D 15

16 The pathogenic variant (2bp deletion 185delAG in exon 3) was directly sequenced in the 5 cell BRCA1-positive sample (5/11 reads, confirming that the deletion was heterozygous) but was not sequenced in the single cell sample. However, analysis of sequence variants in close proximity of the 2bp deletion was possible to enable phasing.

17 +Primers 2: D 3: D 6: Control 7: Control

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19 Click DOPlify to edit TM Workflow Master title style 19 Sample Cell Lysis Whole Genome Amplification DOP-PCR QC step - Agarose Gel Electrophoresis

20 DOPlify Kit Contents 20 Kit Codes Component Cap colour RHS reactions PCR-grade H 2 O White Cell Lysis Enzyme Cell Lysis Buffer WGA Polymerase WGA PCR Buffer Primer Yellow Yellow Red Red Red Store frozen at -20 C

21 Storage Information 21 DOPlify kits are shipped on ice packs. Upon receipt, store the kit at -20 C in a constant temperature freezer (not frost free). Please note that reagents stored at temperatures above -20 C are more prone to degradation and contamination; therefore storage at other temperatures is not recommended. It is not recommended to store reagents at -80 C. When stored under the correct conditions and handled correctly, full activity of the kit is retained until the expiry date indicated on the kit label. This kit is designed to amplify picogram quantities of DNA; therefore extreme caution must be exercised to prevent the introduction of foreign DNA contaminants. Reagents should be stored in a clean laboratory away from potential PCR contaminants. Good laboratory practices for performing PCR should be followed when storing and handling reagents, as well as during the setup of reactions.

22 DOPlify Overview 22 Sample collection o o Place the sample into a PCR tube in <2 µl of buffer 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 Protocol Time (minutes) WGA Hands-on Incubation

23 Input Specifications 23 Template -Single cells or multi-cell aliquots -DNA (up to 30pg/µl) 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)

24 WGA Quality Control 24 The WGA DNA products should appear as a smear ranging in size from 200 bp bp (see WGA Example 1). The no template control (NTC) should appear clean other than the presence of primer dimers 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)

25 WGA Quality Control 25 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 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)

26 DOPlify NGS Application Output data 26 PGS whole chromosome aneuploidy PGS unbalanced translocations/segmental DNA changes SNPs PGD small base deletions Targeted enrichment Mitochondrial DNA load and sequence

27 DOPlify PGS - Single cell aneuploidy 27 Sample: Single cells sorted from cell line of known karyotype (Coriell) 47,XY, Method: 1.80 DOPlify TM single cell WGA Neoprep libraries MiSeq NGS NGS Metric: Chromosome reads per total sample reads, normalized to a reference. Normalised ratio of mapped reads X Y Chromosome

28 DOPlify PGS - Segmental changes 1Mb bins X Y Chromosome

29 DOPlify - SNP detection 29

30 DOPlify PGD 2bp deletion 30 Single Cell 5 Cell gdna Also SNPs for phasing in the absence of sequence coverage

31 DOPlify - Target Sequence Enrichment 31 +Target Specific PCR Primers +Primer Control NTC +Primer +Primer Control Control

32 DOPlify mitochondrial DNA load 32 Sample: Single cells from female individual or cell line of known karyotype (Coriell) Method: DOPlify TM single cell Nextera libraries MiSeq NGS Percent reads (log scale) NGS Metric: Reads mapping to the mtdna genome as percent of total sample reads 0.01 Euploid Aneuploid

33 DOPlify mitochondrial DNA genome 33 PicoPlex PicoPlex DOPlify TM DOPlify TM Mitochondrial DNA sequence analysis of single cells amplified using either DOPlify or PicoPlex (Rubicon). Libraries were prepared using standard Nextera protocol and sequenced in a 23-plex pairedend 150-bp read format on the Illumina NextSeq

34 DOPlify - NGS Library Kit Compatible 34 Illumina Neoprep Nextera TruSeq Thermo Fisher Scientific Ion DNA Fragment Library Kapa Biosystems (Illumina and Ion Torrent) HyperPlus HyperPrep Bioo Scientific (Illumina and Ion Torrent) NEXTflex Rapid DNA-Seq Other: New England BioLabs (Illumina and Ion Torrent) Swift Biosciences (Illumina and Ion Torrent) RHS has undertaken an extensive evaluation of 4 specific NGS library preparation kits data to be released

35 Acknowledgements 35 RHS Team Kimberly Warren Matthew Brockman Christine Robinson Sandra Protopsaltis Rachel Lawson Oksana Mugalimova Ray Ridge Sequencing Collaborators University of NSW (Australia) Garvan Institute (Australia) The Ramaciotti Centre (Australia) SAHMRI (Australia) ACRF (Australia)

36 DOPlify is a Research Use Only product and is not to be used for diagnostic procedures Please contact RHS for ordering information. For more information on the products use, limitations, and licenses: E: info@rhsc.com.au Melinda Jasper melinda.jasper@rhsc.com.au Reproductive Health Science Ltd (ABN ) West Thebarton Road THEBARTON SA AUSTRALIA 5031 Ph: ASX:RHS Reproductive Health Science Ltd