This document discusses the use of next generation sequencing (NGS) technology for preimplantation genetic screening (PGS). It provides background on PGS and describes the timeline of technologies used, including fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH), SNP microarrays, and NGS. VeriSeq PGS is highlighted as an NGS assay developed by Illumina for PGS using embryos biopsied on day 3 or 5. Studies validating VeriSeq showed 100% consistency with aCGH and karyotyping in detecting chromosome abnormalities. NGS provides advantages over other methods like higher resolution, batch analysis to reduce costs, and ability to detect mosaicism.

1. NGS: The New Technology in
Preimplantation Genetic Screening
Methee Sriprapun (MT, PhD)
Postdoctural Research Fellow
Research Unit of Hepatitis and Liver Cancer, Department of Biochemistry
Faculty of Medicine, Chulalongkorn University

2. Application of NGS
in Preimplantation
Genetic Screening

3. Content
•Background of preimplantation genetic screening
(PGS)
•Timeline of PGS Technology
•Next generation sequencing in PGS (VeriSeqTM)

4. 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

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

6. 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

7. Dahdouh EM, et al. (2009)
Pros & Cons of different biopsy
specimens

8. 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. 13
Chr. 18
Chr. 21
Chr. X
Chr. Y
Harton GL, et al. (2011)

9. 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

10. 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)

11. 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

12. Timeline of PGS technology (5)
Next generation sequencing (NGS)
http://assets.illumina.com/content/dam/illumina-marketing/images/rgh/MiSeq.jpg

13. •High-throughput DNA sequencing technology
•Parallel whole genome sequencing
•Developed instead of Sanger sequencing
•Principle of each NGS technology depending on
each machine
NGS

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

15. 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

16. •Luanched by Illumina in 2014 for working with Illumina's
NextSeqTM 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

17. 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

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

19. Ref: Illumina data sheet

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

21. VeriSeqTM PGS Workflow
Ref: Illumina data sheet

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

23. BlueFuse Multi Analysis Software
Ref: Illumina data sheet
Experiment
information
Sample profile
Decision track information
Karyotype chart
Report

24. Report from NGS-PGS
Ref: Illumina data sheet

25. Ref: Illumina data sheet

26. Ref: Illumina data sheet

27. NGS-PGD improved pregnancy and implantation
rates when analyzing with blastomeres (D3)
Sequencing with Ion
Personal Genome
Machine®
(PGM™) System
https://www.thermofisher.com

28. Objective
To validate NGS for 24-chromosome aneuploidy
screening 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

29. 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

30. Findings
Consistency results of D3 biopsy
between 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%

31. 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

32. Hiseq instrument
Miseq instrument
Consistency results between Hiseq and Miseq instruments

33. False positive of aneuploidy screening with NGS
aCGH
NGS Trisomy 18
False Positive
result

34. 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

35. Objective
Sample recruitment
To investigate the accuracy of NGS technology for comprehensive
chromosome screening and aneuploidy detection at blastocyst stage
(D5-D6 biopsy)
Trophectoderm (TE) biopsy samples and cytogenetically characterized
cell lines (Coriell Cell Repositories)

36. 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 TE
2. Partial deletion and duplication validated with Coriell cell line
3. Microdeletion could not be detected from both methods

38. Results of copy number changes
aCGH NGS
Monosomy X
Monosomy 4,5,18 and 19
Trisomy 11,14,22 and monosomy 19

39. 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

40. •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

41. Comparison of NGS and other
techniques
https://www.progenesis.com/why-ngs-24/

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

43. Comparison of all 24-chromosome
copy number analysis
Handyside AH (2013)

44. Thank you for your
attention