NGS: The New Technology in

Preimplantation Genetic Screening

Methee Sriprapun (MT, PhD)

Postdoctural Research Fellow

Research Unit of Hepatitis and Liver Cancer, Department of BiochemistryFaculty of Medicine, Chulalongkorn University

Application of NGS in PreimplantationGenetic Screening

Content

•Background of preimplantation genetic screening

(PGS)

•Timeline of PGS Technology

•Next generation sequencing in PGS (VeriSeqTM)

Preimplantation Genetic Screening (PGS)

“Genetic methods to screening

chromosomal abnormalities in each

embryo and select the most competent

embryo to transfer to mother”

Purpose:

Palini S, et al. (2015)

To improve the success rate of ET and pregnancy

Ref: PowerPoint Slides of LaTasha B. Craig downloaded from www.slideshare.net

Specimens for PGS testing

Embryo biopsy at Day 3 (1-2 cells of each blastomere)

Embryo biopsy at Day 5-6 (blastocyst stage)

Trophectoderm or polar body biopsy

http://www.stork.co.th/en/our-services/treatment-types/embryo-biopsy-prior-to-pgd

Dahdouh EM, et al. (2009)

Pros & Cons of different biopsy specimens

Timeline of PGS technology

Fluorescence In Situ Hybridization (FISH)

http://www.viagenefertility.com/photos/fish-male.jpg

• First molecular technique in PGS/PGD• Limitation in No. of investigated chromosomes • Problem: non-specific hybridization, Signal interpretation

PGS FISH during D3 not improving Pregnancy rate

Chr. 13Chr. 18Chr. 21Chr. XChr. Y

Harton GL, et al. (2011)

Timeline of PGS technology (2)

Array Comparative Genomic Hybridization (aCGH)

https://www.dovepress.com/cr_data/article_fulltext/s53000/53422/img/fig7.jpg

• First accurate and fast technology for PGS (12-24 hrs)• Reduce cost per sample due to increasing of processed

sample simultaneously• 24 Chromosome analysis

Timeline of PGS technology (3)

SNP microarray

qPCR

• 24 chromosome analysis• Duplication and deletion detection• Chromosomal translocation detection• Determine the intensity ratio of 2 SNP alleles

at heterozygous loci• Require father and mother genotypes

• Chromosome copy analysis• 24 chromosome analysis• Suitable for multiple cells such as TE biopsy (D5-D6)

Palini S, et al. (2015) and Dahdouh EM, et al. (2009)

Timeline of PGS technology (4)

The KaryoLite™ BACs-on-Beads™ (KL-BoBs™)

“24 chromosome detection using amplification and hybridization methods”

*Rapid analysis of aneuploidies in all 24 chromosomes*

http://www.wellconn.com/imgs/karyolite.gif

Timeline of PGS technology (5)

Next generation sequencing (NGS)

http://assets.illumina.com/content/dam/illumina-marketing/images/rgh/MiSeq.jpg

•High-throughput DNA sequencing technology

•Parallel whole genome sequencing

•Developed instead of Sanger sequencing

•Principle of each NGS technology depending on

each machine

NGS

http://www.slideshare.net/ueb52/introduction-to-next-generation-sequencing-v2

NGS in routine PGS

Why NGS is validated in PGS technology ?

• High-throughput and high resolution technology

• Clearly defined mosaicism in embryo biopsy

• High accuracy and sensitivity for PGS

• Batch analysis reduce cost per test

• Be applied for non-invasive prenatal testing

Fiorentino F et al. (2014); Zheng et al. (2015); VeriSeqTM illumine sheet

• Luanched by Illumina in 2014 for working with Illumina'sNextSeqTM 500 and MiSeq® sequencing systems

•Using at least 1 ng of amplified DNA from D3 or D5 embryo biopsy

•Analyzing results with BlueFuse Multi analysis software

•Completed processing method in 12 hours

VeriSeqTM PGS assay

http://www.illumina.com/products/veriseq-pgs.html

MiSeq® sequencing machine

http://assets.illumina.com/content/dam/illuminamarketing/images/landing/iswitched/miseq_iswitch.jpg

NextSeqTM 500 sequencing machine

http://assets.illumina.com/content/dam/illuminamarketing/images/systems/nextseq/nextseq-large.jpg

MiSeq Technology

“Sequencing by synthesis (SBS) technology”http://bitesizebio.com/13546/sequencing-by-synthesis-explaining-the-illumina-sequencing-technology/

Ref: Illumina data sheet

http://www.slideshare.net/Research_Instruments/new-solutions-for-genetic-testing-of-embryos

VeriSeqTM PGS Workflow

Ref: Illumina data sheet

http://www.slideshare.net/Research_Instruments/new-solutions-for-genetic-testing-of-embryos

BlueFuse Multi Analysis SoftwareRef: Illumina data sheet

Experimentinformation

Sample profile

Decision track information

Karyotype chart

Report

Report from NGS-PGSRef: Illumina data sheet

Ref: Illumina data sheet

Ref: Illumina data sheet

NGS-PGD improved pregnancy and implantationrates when analyzing with blastomeres (D3)

Sequencing with Ion Personal Genome Machine®(PGM™) System

https://www.thermofisher.com

Objective

To validate NGS for 24-chromosome aneuploidyscreening and To investigate the applicability to PGS

Sample recruitment

1. Single cells with known chromosomal abnormalities by karyotyping method

2. D3 WGA product from previously analyzed with aCGH

NGS analysis (2)

1. MiSeq (Illumina) and performing alignment using Bwa tool (MiSeq reporter software

2. Filtering and analysis with the same programs as HiSeq

NGS analysis

1. Hiseq 2000 (Illumina) and sequence analysis with iSAAC(Hiseq analysis software) and Bluefuse software

2. Remove unmapped, duplicated and low mapping reads by BEDtools and SAMtools

Findings

Consistency results of D3 biopsybetween NGS and either

conventional karyotyping or aCGH assay

Specificity of NGS (consistency with copy No. of chromosome) 99.98%Sensitivity of NGS (consistency with copy No. of chromosome) 100.00%

Specificity of NGS (24-chromosome diagnosis consistency) 100%Sensitivity of NGS (24-chromosome diagnosis consistency) 100%

aCGH NGS

Monosomy 9 Monosomy 9

Monosomy 7, 18; trisomy 16 Monosomy 7, 18; trisomy 16

Results of copy number changes

Trisomy 2,7,9,10,19,21,22; monosomy 5, 13,X

Trisomy 2,7,9,10,19,21,22; monosomy 5, 13,X

Hiseq instrument

Miseq instrument

Consistency results between Hiseq and Miseq instruments

False positive of aneuploidy screening with NGS

aCGH

NGS Trisomy 18

False Positive result

aCGH NGS

Results of partial aneusomy detection

14 Mb segmental duplication of 17p

20 Mb segmental gain of 13q

17 Mb segmental duplication of 7p

Objective

Sample recruitment

To investigate the accuracy of NGS technology for comprehensivechromosome screening and aneuploidy detection at blastocyst stage (D5-D6 biopsy)

Trophectoderm (TE) biopsy samples and cytogenetically characterizedcell lines (Coriell Cell Repositories)

Test methodology

WGA product was assessed with either aCGH and VeriSeq NGS (MiSeq)

Findings

Consistency results between aCGH and NGS in chromosome abnormalities:

1. Chromosome copy number variations (loss & gain) validated with TE2. Partial deletion and duplication validated with Coriell cell line 3. Microdeletion could not be detected from both methods

Results of copy number changes

aCGH NGS

Monosomy X

Monosomy 4,5,18 and 19

Trisomy 11,14,22 and monosomy 19

Results of partial aneusomy detection

aCGH NGS

2.19 Mb segmental duplication of 6p

2.53 Mb segmental deletion of 5q

1.81 Mb segmental deletion of 9p

•Most accurate and informative

•Sequence data from thousands of loci along chromosome

•Multiple genomic loci and multiple samples on one chip (aCGH: 1 sample/chip)

•Need to be further validated in various cohort

Advantages of NGS

Comparison of NGS and other techniques

https://www.progenesis.com/why-ngs-24/

Ref: PowerPoint Slides of Dmytro Mykytenko downloaded from www.slideshare.net

Comparison of all 24-chromosome copy number analysis

Handyside AH (2013)

Thank you for your attention