(PGS) for Aneuploidy and Haplotyping of the ANXA5 Gene

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Combined pre-implantation genetic screening (PGS) for aneuploidy and haplotyping of the ANXA5 gene Screening for chromosomal aneuploidy and predisposition to miscarriage in IVF embryos in real time means no freeze/thawing and reduces the waiting time for embryo transplantation Contact: [email protected] More information at: www.nanoporetech.com and publications.nanoporetech.com

Biopsy of gDNA Whole-genome a) Day 1 Day 3 Day 5 exon 2 exon 1 1–3 cells extraction amplification ANXA5-001 ANXA5-003 long amplicon for blood gDNA short amplicon for WGA DNA insertion deletion SNVs M2 SNVs

F PCR with specific primers b) 100 R for one or more genes Haplotype G-A-T-G (WT) 80 Simultaneous end-prep of ANXA5 amplicon A-C-C-A (M2) and accompanying WGA DNA p A 60 A p A p p A Attachment of native barcode, adapter and tether 40

% of each haplotype 20

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Sequencing Sample

Fig. 1 Workflow for combined aneuploidy screening and single-locus testing Fig. 2 ANXA5 a) locus and variants b) haplotyping results. Blue = wild-type, green = M2 PGS and PGD improve IVF success rate by ANXA5 M2 haplotype is associated with screening for abnormalities increased risk of miscarriage PGS is the process of screening an IVF embryo for aneuploidy by counting chromosome The human ANXA5 gene, situated on chromosome 4, encodes a calcium-dependent number, using low-coverage sequencing of the whole genome. Conversely, preimplantation phospholipid binding protein, which acts as a placental anticoagulant. A variant haplotype of genetic diagnosis (PGD) tests a single gene, and requires higher coverage of that region, for ANXA5 contains 4 nucleotide substitutions which lie within the space of 57 nucleotides in the variant-calling. PCR amplification of the single region for a limited number of cycles enriches for promoter (Fig. 2a). These substitutions reduce the activity of the promoter substantially, and if the the target region, without overwhelming the whole-genome template. Following amplification, embryo inherits the M2 haplotype from either parent, the risk of miscarriage increases sequencing adapters can be attached to both the amplicon and the accompanying substantially. Rather than testing the parents, by amplifying across the region in blastocyst DNA, whole-genome template, creating a combined PGS-PGD sequencing library (Fig. 1). followed by sequencing, we are able to identify embryonic ANXA5 haplotypes (Fig. 2b).

a) Increasing resolution 1 1 100 2 2 100 80 3 3 500 4 4 5 5 1,000 60 6 6 7 7 5,000 40

8 8 Accuracy

Bin width (kb) 10,000 9 9 20 10 10 11 11 20,000 12 12 0 13 13 5,000 10,000 50,000 100,000 14 14 Reads 15 15 b) Chromosome Chromosome Chromosome 16 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2122 X Y 17 17 4 18 18 3 19 19 2 20 20 1 21 21 Coverage 0 22 22 X X Y Y 1 2 3 4 5 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0 0.5 1.0 1.5 2.0 2.5 3.0 4 3 Ploidy level 47, XX, +16 Ploidy level 44, XY, -15, -16 Sample: ONT34 Sample: ONT39 2 ANXA5 diplotype GATG (WT), homozygote GATG (WT) 1

ANXA5 diplotype Coverage 0 ACCA (M2)

Fig. 3 Results of combined PGS and ANXA5 haplotyping in two aneuploid embryos Fig. 4 Higher-resolution PGS screening a) read depth and resolution b) Cri du chat syndrome Combined aneuploidy screening and ANXA5 Aneuploidy and higher-resolution analyses haplotyping of blastocyst DNA from low-coverage nanopore data We extracted genomic DNA from 1–3 cells taken from thirty 5-day-old IVF blastocysts and We took a higher-coverage PGS dataset and downsampled the data, to find the minimal number performed whole genome amplification (WGA) of the DNA. Combined PGS/ANXA5 libraries were of reads required to robustly identify the ploidy level of a sample. We calculated that 50,000 created and the barcoded products were quantified before being pooled and sequenced, 6 reads of 500 nt in length are required, equating to approximately 0.01x, or 30 Mb, per sample samples per flowcell. Sequence reads were analysed using our PGS bioinformatics pipeline and (Fig. 4a). Our calculations also indicate that without significantly greater coverage, we can detect results of both aneuploidy screening and ANXA5 haplotyping for two samples are shown in Fig. smaller-scale abnormalities than whole-chromosome aneuploidies. To test this, we prepared the 3. Our ploidy calls for each sample were in full concordance with the CGH results, and the same library type from a cell line carrying the Cri du Chat deletion. The partial deletion of the ANXA5 haplotype calls of each sample were verified by capillary sequencing. short arm of chromosome 5 is clearly visible, along with several cell-line artefacts (Fig. 4b).