Rapid genomic screening of embryos using nanopore sequencing

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1 Rapid genomic screening of embryos using nanopore sequencing Daniel J Turner, PhD Senior Director of Applications Oxford Nanopore Technologies Forman EJ & Scott RT Jr Contemporary OB/GYN () 2014 Euploid single-embryo transfer: the new IVF paradigm?

2 What is a nanopore? Deamer & Branton et al., ~20 years ago - Nanopore Membrane + Translocation 10 ~ nm Protein nanopores occur naturally in cells and can embed themselves in membranes Nanopores create tiny holes or channels a few nanometers in diameter Molecules can pass from one side of a membrane to the other Deamer s notebook

3 How does nanopore sequencing work? - Axopatch 200B - single channel patch clamp amplifier + Nanopore translocation Open current Red = current change 1 Purple = current change 2

1,500 ~ nt transcript - 150 100 Nanopore translocation 50 0 5 10 15

4 Current (pa) How does nanopore sequencing work? open pore current open pore current adapter polya tail 1,500 ~ nt transcript Nanopore translocation Time (sec) Open current Red = current change 1 Purple = current change 2 RNA squiggle +

5 How does nanopore sequencing work? Consumable flow cell Sample port Sensor chip: what s inside: Biological nanopore Sensing channel Array of channels Motor Nanopore Membrane USB powers device and passes data to PC Connector flow cell connects with MinION Electronic chip

motor stall 1. Long reads: tens to hundreds of kilobases 2. Portability 3.

6 How does nanopore sequencing work? adapter DNA of interest adapter leader strand tether oligo motor protein motor stall 1. Long reads: tens to hundreds of kilobases 2. Portability 3. Low cost 4. Data generation and analysis in real time A. Run-until (no sample batching necessary) B. Read-until

Nanopore Applications Sample extraction &

7 Nanopore Applications Sample extraction & library preparation Use-cases / showcasing Bioinformatics Pathogens Environment Microbiome Basic genome Transcriptomics research Human Cancer Clinical Plant Population genetics research research research genomics

8 Detecting aneuploidy with high-throughput sequencing Day 1 Day 3 Day 5 Biopsy of 1 3 cells gdna extraction Library preparation and sequencing Coverage normalised to haploid reference Coverage normalised to haploid reference Euploid male Male with trisomy 13

9 Why do PGS / PGD with nanopores? Programme start: Hormone Egg Blastocyst Embryo freezing / PGS test diagnostic tests, stimulation collection biopsy vitrification medical visits 4 3weeks 9 7days After 5 days 14~ days Patient s next cycle Embryo Embryo Medical supervision thawing transfer over implantation

10 Why do PGS / PGD with nanopores? Programme start: Hormone Egg Blastocyst Embryo freezing / PGS test diagnostic tests, stimulation collection biopsy vitrification medical visits 4 3weeks 9 7days After 5 days 14~ days Same cycle 1. Results faster 2. Lower cost per sample 3. Control over whole process Embryo Embryo Medical supervision 4. Lower startup cost thawing transfer over implantation

heparin daily Testing embryo would stop unnecessary treatment 100 80 60 G-A-T-G (WT) A-C-C-A (M) 2 40 20 0 1 2 3 4 5 6 7 8 9 10

11 %of each haplotype ANXA5 ANXA5-001 ANXA5-003 long amplicon for blood gdna short amplicon for WGA DNA insertion deletion SNVs M2 SNVs exon 2 exon 1 Placental anticoagulant M2 haplotype associated with recurrent miscarriage M2 parent > expectant mother given heparin daily Testing embryo would stop unnecessary treatment G-A-T-G (WT) A-C-C-A (M) Sample PCR and nanopore sequencing identifies M2 haplotype Results confirmed by capillary sequencing

screen Effective but inconvenient PCR with specific primers End-prep of WGA DNA End-prep of ANXA5 amplicon

12 Combined aneuploidy screen and ANXA5 haplotyping Biopsy of 1 3 cells gdna extraction Whole genome amplification WGA sample divided into 2 One half used for ANXA5 haplotying assay One half used for aneuploidy screen Effective but inconvenient PCR with specific primers End-prep of WGA DNA End-prep of ANXA5 amplicon Adapter and tether attachment Adapter and tether attachment Sequencing 2017 Oxford Nanopore Technologies. All rights reserved.

Combined aneuploidy screen and ANXA5 haplotyping Biopsy of 1 3 cells gdna extraction Whole genome amplification WGA sample divided into 2 One half used for ANXA5 haplotying assay One half used for

13 Combined aneuploidy screen and ANXA5 haplotyping Biopsy of 1 3 cells gdna extraction Whole genome amplification WGA sample divided into 2 One half used for ANXA5 haplotying assay One half used for aneuploidy screen Effective but inconvenient Combined assay uses limited number of PCR cycles PCR with specific primers Simultaneous end-prep of ANXA5 amplicon and accompanying WGA DNA Adapter and tether attachment Sufficient WGA DNA is still present at the end of PCR All DNA is prepared for sequencing Low-coverage, whole-genome data and highercoverage amplicon data are generated together Easily adapted to other genes (e.g. Huntingtin) Sequencing 2017 Oxford Nanopore Technologies. All rights reserved.

Combined aneuploidy screen and ANXA5 haplotyping Coverage normalised to haploid reference Coverage normalised to haploid reference Sample: ONT34

heterozygote All ANXA5 haplotypes confirmed by capillary All ploidy levels confirmed by aray-cgh Specific region / regions at higher coverage

14 Combined aneuploidy screen and ANXA5 haplotyping Coverage normalised to haploid reference Coverage normalised to haploid reference Sample: ONT34 Ploidy level ANXA5 diplotype, 47XX, +16 GATG (WT), homozygote Sample: ONT39 Ploidy level ANXA5 diplotype, 44XY, -14, -16 GATG (WT), ACCA (M2) heterozygote All ANXA5 haplotypes confirmed by capillary All ploidy levels confirmed by aray-cgh Specific region / regions at higher coverage for SNPs Long reads would allow amplicons of several kb Whole genome at low coverage for ploidy 2017 Oxford Nanopore Technologies. All rights reserved.

15 Bin width (kb) Accuracy Coverage - how low can we go? , ,000 10,000 20, ,000 10,000 50, ,000 Number of reads Ability to call aneuploidies robustly from low coverage: 50,000 reads, 500 nt in length required per sample = ~ 0.01x Can either multiplex to get low cost per sample, or use run-until to get results very quickly Cheaper 128-channel flowcells will be available imminently

Bin width (kb) Accuracy Next steps Increasing resolution 100 500 100 80 Wild-type 20 10kb, 95% ~ homology Distal

Proximal 5,000 10,000 50,000 100,000 Number of reads No significant increase in coverage required to detect

16 Bin width (kb) Accuracy Next steps Increasing resolution Wild-type 20 10kb, 95% ~ homology Distal Dosage-sensitive gene / genes Proximal Centromere 1, , Deletion 10,000 20, Duplication Distal Proximal 5,000 10,000 50, ,000 Number of reads No significant increase in coverage required to detect sub-chromosomal changes Microdeletions and duplications are 1 5 Mb and can arise by de novo NAHR events during meiosis Consequences can be devastating

17 Acknowledgements Care Fertility Oxford Nanopore Technologies Simon Fishel Sissel Juul Eoghan Harrington Applications team All ONT staff

Targeted qpcr. Debate on PGS Technology: Targeted vs. Whole genome approach. Discolsure Stake shareholder of GENETYX S.R.L

Targeted qpcr. Debate on PGS Technology: Targeted vs. Whole genome approach. Discolsure Stake shareholder of GENETYX S.R.L Antonio Capalbo, PhD Laboratory Director GENETYX, reproductive genetics laboratory, Italy PGT responsible GENERA centers for reproductive medicine, Italy Debate on PGS Technology: Targeted vs. Whole genome