Preimplantation Genetic Testing for Monogenic/Single Gene Disoders (PGT-M, PGD) Luis A. Alcaraz
Disclaimer Thermo Fisher Scientific and its affiliates are not endorsing, recommending, or promoting any use or application of Thermo Fisher Scientific products presented by third parties during this seminar. Information and materials presented or provided by third parties are provided as-is and without warranty of any kind, including regarding intellectual property rights and reported results. Parties presenting images, text and material represent they have the rights to do so. Speaker was provided travel and hotel support by Thermo Fisher Scientific for this presentation, but no remuneration.
Simplified Workflow PGT-A A + M Is PGT-SR 1 HR-CGH Big? Easy to combine in one single protocol. Is Is BIOPSY BIOPSY REPROSEQ Structural? Structural? Normal Abnormal Yes No No Risk reduc ducti tion 2 Normal Abnormal PGT-A PGT-M PGT-M Less sample handling. No No Is Point Is Point D-3 D-5 Mutation? Mutation? Biopsy Biopsy Cost st reduc ducti tion on 3 Yes Yes miniSeq miniSeq aCGH Less investment in equipment and human resources. Direct Direct aCGH Normal Normal NGS Test Test Afforda ordable le Normal Abnormal 4 Indirect Indirect Abnormal Abnormal STRs STRs Test Test Any patient can opt.
Direct Di ect Test In Indire irect ct Test st Linkage analysis Mutation analysis Needed in any case. Relatives are needed Not always possible due to (informativity test) technical limitations. Analysis of several polymorphisms simultaneously High risk of allele dop-out. at both sides of the gene Usually is done with mini- Low risk of allele drop-out. sequenicng Low risk of recombination. Usually by STR Direct and Indirect Test When possible, both tests are done simultaneously, however, the final result relies always in the indirect test.
Allele Dropout Both alleles are amplified. One allele is amplified: FALSE NEGATIVE.
PGT-M by NGS 2 Mb 2 Mb ThermoFisher
What is? 1 Reagent t Kit Includes all the reagents needed for PGT-M library prep, including primer pool, Ampliseq reagents and barcodes. 2 Software re An Analysi ysis Includes access to the analysis software for free, with support from one of our FAS. 3 For PGT-M For the analysis of single gene disorders when the parents are carriers. Based on linkage analysis of dozens of SNPs to identify parental mutated/healthy alleles. 4 With PGT-A Combinable with Reproseq in one single workflow.
Lab Protocol 3 ul PGT-A PGT-M Template Sequencing Analysis Ion Ion 100 0 bp bp PGD-Seq IA IA ReproSeq roSeq Reporte rter 4 h 4 h 1 h 2.5 h 2 h
SNP Phasing A/T T/T A T T T A/T A T
SNP Phasing A/T T/T A T T T A/T A/T A/T T/T T/T A T A T A T T T T T
SNP Phasing C/G C/C A T T T C G C C C/C C/C C/C G/C G/C A T A T A T T T T T C C C C C C G C G C
SNP Phasing G/G T/G A T T T C G C C G G T G G/G G/G G/T G/G G/T A T A T A T T T T T C C C C C C G C G C G G G G T G G G T G
SNP Phasing C/C C/A A T T T C G C C G G T G C C C A C/A C/A C/C C/A C/C A T A T A T T T T T C C C C C C G C G C G G G G T G G G T G C A C A C C C A C C
SNP Phasing A T T T C G C C G G T G C C C A A T A T A T T T T T C C C C C C G C G C G G G G T G G G T G C A C A C C C A C C
SNP Phasing A T T T C G C C G G T G C C C A Affected Carrier Carrier Normal A T A T A T T T T T C C C C C C G C G C G G G G T G G G T G C A C A C C C A C C
Analysis Protocol
SNP Phasing
SNP Phasing
SNP Phasing
SNP Phasing
Analysis Software Reads VCF file from the sequencer. Performs linkeage analysis with parental and relatives samples. Linkeage analysis to embryos Supervised by our Support Team. PGD-Seq software analysis is offered for free. It performs linkeage analysis to the parental samples, using a child for SNPS phasing. Finally, embryos are analyzed and affected/healthy alleles are identified.
PGD-Seq Examples BRCA2 :c.15 :c.1597d 97delA lA (p.Th Thr533 533Le Leuf ufsX25 X25) nd Cycle st Cycle 1 st 2 nd cle (8 8 embryo ryos) s) cle (10 0 embr bryo yos) s) Non carrier Non carrier 6 7 Euploid Euploid 5 5 Healthy embryos Healthy embryos 4 3
PGD-Seq Validation PKD1 :c.7292T>A (p.Leu2431Gln) Perf rforman rmance ce
PGD-Seq Validation PKD1 :c.7292T>A (p.Leu2431Gln) Perf rforman rmance ce Full ll conco ncorda rdance nce
Balanced Translocations Balanced Normal
How it is Used The kit is ordered according to PGT-M case (gene) and Orderin ering delivered. Sequencing and analysisof familial samples is first performed PGT-M Set up for PGT-M setup. IVF cycle begins and resulting embryos are eventually IVF Cycle biopsied. Amplifi ificat catio ion Biopsies are amplified and sequenced. Embryo results are analyzed via software and validated An Analysis for final report.
PGD-Seq Features Based d on SNPs Based d on NGS SNP linkage provides more robust analysis to avoid The most efficient technology for DNA allele drop-out analysis Flexible le Extensive sively ly Validated dated Easy to customize to any need Validated in clinical use with 150 different monogenic disorders Support t Center ter Simplifi fied workflo flow Our support center assists from case preparation Simplifies the laboratory routine even allowing to data analysis fresh transfers
Platform Comparison Ion ReproSeq roSeq Karyo ryomapping VeriSeq PGD-Seq Aneuploidy uploidy screening ng Detecti tion on of poliploidy ploidy Detecti tion on of segmenta ntal aberrat ration ons Detecion on of mitotic otic error ors (mos osai aicism) Indire rect t Test Direct t Test (poi oint t muta tati tion on) Fresh transfer Scala alable ble Price
Acknowledgments Santiago González-Reig Vanessa Penacho Marta Alfonso Diego Amorós Andrés Antón Helena Blanca Cristina Botella Tatiana Garijo Rubén Dasí Francisco Galán Alba López Irene Manchón-Trives Paula Brígido Alain Rico Melanie Haessler Miguel Fernández Helena Blanca Santiago González-Reig Santiago González
luis.alcaraz@bioarray.es http://www.bioarray.es Thank you!
Recommend
More recommend
