Combined pre-implantation genetic screening (PGS) for aneuploidy and haplotyping of the ANXA5 geneScreening 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

Fig. 1 Workflow for combined aneuploidy screening and single-locus testing

The human ANXA5 gene, situated on chromosome 4, encodes a calcium-dependent phospholipid binding protein, which acts as a placental anticoagulant. A variant haplotype of ANXA5 contains 4 nucleotide substitutions which lie within the space of 57 nucleotides in the promoter (Fig. 2a). These substitutions reduce the activity of the promoter substantially, and if the embryo inherits the M2 haplotype from either parent, the risk of miscarriage increases substantially. Rather than testing the parents, by amplifying across the region in blastocyst DNA, followed by sequencing, we are able to identify embryonic ANXA5 haplotypes (Fig. 2b).

PGS and PGD improve IVF success rate by screening for abnormalities

Combined aneuploidy screening and ANXA5 haplotyping of blastocyst DNA

Fig. 2 ANXA5 a) locus and variants b) haplotyping results. Blue = wild-type, green = M2

© 2017 Oxford Nanopore Technologies. All rights reserved.P17005 - Version 4.0

Fig. 3 Results of combined PGS and ANXA5 haplotyping in two aneuploid embryos

We extracted genomic DNA from 1–3 cells taken from thirty 5-day-old IVF blastocysts and performed whole genome amplification (WGA) of the DNA. Combined PGS/ANXA5 libraries were created and the barcoded products were quantified before being pooled and sequenced, 6 samples per flowcell. Sequence reads were analysed using our PGS bioinformatics pipeline and results of both aneuploidy screening and ANXA5 haplotyping for two samples are shown in Fig. 3. Our ploidy calls for each sample were in full concordance with the CGH results, and the ANXA5 haplotype calls of each sample were verified by capillary sequencing.

We took a higher-coverage PGS dataset and downsampled the data, to find the minimal number of reads required to robustly identify the ploidy level of a sample. We calculated that 50,000 reads of 500 nt in length are required, equating to approximately 0.01x, or 30 Mb, per sample (Fig. 4a). Our calculations also indicate that without significantly greater coverage, we can detect smaller-scale abnormalities than whole-chromosome aneuploidies. To test this, we prepared the same library type from a cell line carrying the Cri du Chat deletion. The partial deletion of the short arm of chromosome 5 is clearly visible, along with several cell-line artefacts (Fig. 4b).

Aneuploidy and higher-resolution analyses from low-coverage nanopore data

Fig. 4 Higher-resolution PGS screening a) read depth and resolution b) Cri du chat syndrome

Sequencing

Attachment of native barcode, adapter and tether

Simultaneous end-prep of ANXA5 ampliconand accompanying WGA DNA

pA

Ap

b)

a)

ANXA5 M2 haplotype is associated with increased risk of miscarriage

PGS is the process of screening an IVF embryo for aneuploidy by counting chromosome number, using low-coverage sequencing of the whole genome. Conversely, preimplantation genetic diagnosis (PGD) tests a single gene, and requires higher coverage of that region, for variant-calling. PCR amplification of the single region for a limited number of cycles enriches for the target region, without overwhelming the whole-genome template. Following amplification, sequencing adapters can be attached to both the amplicon and the accompanying whole-genome template, creating a combined PGS-PGD sequencing library (Fig. 1).

% o

f eac

h ha

plot

ype

G-A-T-G (WT)A-C-C-A (M2)

Haplotype

gDNA extraction

Biopsy of 1–3 cellsDay 5Day 3Day 1 Whole-genome

amplificationexon 2 exon 1

M2 SNVsSNVs

deletioninsertion

ANXA5-001ANXA5-003

long amplicon for blood gDNAshort amplicon for WGA DNA

100

80

60

40

20

0

Sample1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

PCR with specific primersfor one or more genes

FR

pAp A

0.50 0

123456789

10111213141516171819202122XY

123456789

10111213141516171819202122XY

1.0 1.5 2.0 2.5

Chr

omos

ome

Chr

omos

ome

3.0 3.5 0.5 1.0 1.5 2.0 2.5 3.0

Sample: ONT34Ploidy levelANXA5 diplotype

47, XX, +16GATG (WT), homozygote

Sample: ONT39Ploidy level

ANXA5 diplotype

44, XY, -15, -16GATG (WT)ACCA (M2)

Reads

Chromosome

Increasing resolution

Accu

racy

100

80

60

40

20

0

100

41 2 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2122 X Y3

1 2 4 53

3210

Bin

wid

th (k

b)

Cove

rage

3210

4

Cove

rage

500

1,000

5,000

10,000

20,000

5,000 10,000 50,000 100,000

Contact: publications@nanoporetech.com More information at: www.nanoporetech.com and publications.nanoporetech.com

a)

b)