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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