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Next generation sequencing in preimplantation genetic screening (NGS in PGS)


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Summary trends of NGS in PGS

Published in: Science
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Next generation sequencing in preimplantation genetic screening (NGS in PGS)

  1. 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. 2. Application of NGS in Preimplantation Genetic Screening
  3. 3. Content •Background of preimplantation genetic screening (PGS) •Timeline of PGS Technology •Next generation sequencing in PGS (VeriSeqTM)
  4. 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. 5. Ref: PowerPoint Slides of LaTasha B. Craig downloaded from
  6. 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
  7. 7. Dahdouh EM, et al. (2009) Pros & Cons of different biopsy specimens
  8. 8. Timeline of PGS technology Fluorescence In Situ Hybridization (FISH) • 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. 9. Timeline of PGS technology (2) Array Comparative Genomic Hybridization (aCGH) • 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. 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. 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*
  12. 12. Timeline of PGS technology (5) Next generation sequencing (NGS)
  13. 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. 14.
  15. 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. 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
  17. 17. MiSeq® sequencing machine NextSeqTM 500 sequencing machine
  18. 18. MiSeq Technology “Sequencing by synthesis (SBS) technology”
  19. 19. Ref: Illumina data sheet
  20. 20.
  21. 21. VeriSeqTM PGS Workflow Ref: Illumina data sheet
  22. 22.
  23. 23. BlueFuse Multi Analysis Software Ref: Illumina data sheet Experiment information Sample profile Decision track information Karyotype chart Report
  24. 24. Report from NGS-PGS Ref: Illumina data sheet
  25. 25. Ref: Illumina data sheet
  26. 26. Ref: Illumina data sheet
  27. 27. NGS-PGD improved pregnancy and implantation rates when analyzing with blastomeres (D3) Sequencing with Ion Personal Genome Machine® (PGM™) System
  28. 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. 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. 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. 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. 32. Hiseq instrument Miseq instrument Consistency results between Hiseq and Miseq instruments
  33. 33. False positive of aneuploidy screening with NGS aCGH NGS Trisomy 18 False Positive result
  34. 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. 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. 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
  37. 37. Results of copy number changes aCGH NGS Monosomy X Monosomy 4,5,18 and 19 Trisomy 11,14,22 and monosomy 19
  38. 38. 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
  39. 39. •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
  40. 40. Comparison of NGS and other techniques
  41. 41. Ref: PowerPoint Slides of Dmytro Mykytenko downloaded from
  42. 42. Comparison of all 24-chromosome copy number analysis Handyside AH (2013)
  43. 43. Thank you for your attention