Testing for Monogenic/Single Gene Disoders (PGT-M, PGD) Luis A. - - PowerPoint PPT Presentation

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

Easy to combine in one single protocol.

1 PGT-A A + M

Less sample handling.

2 Risk reduc ducti tion

Less investment in equipment and human resources.

3 Cost st reduc ducti tion

• n

Any patient can opt.

4 Afforda

• rdable

le

PGT-SR Indirect Test STRs Is Structural? D-5 Biopsy D-3 Biopsy aCGH aCGH NGS Normal Abnormal Normal Abnormal Is Big? HR-CGH Normal Abnormal BIOPSY Is Point Mutation? PGT-A Yes PGT-M No Direct Test miniSeq Yes No Indirect Test STRs Is Structural? Normal Abnormal REPROSEQ Normal Abnormal BIOPSY Is Point Mutation? PGT-M No Direct Test miniSeq Yes No

Direct and Indirect Test

Di Direct ect Test In Indire irect ct Test st

Not always possible due to technical limitations. High risk of allele dop-out. Usually is done with mini- sequenicng When possible, both tests are done simultaneously, however, the final result relies always in the indirect test. Mutation analysis Linkage analysis Needed in any case. Analysis of several polymorphisms simultaneously at both sides of the gene Low risk of allele drop-out. Low risk of recombination. Usually by STR Relatives are needed (informativity 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

Includes all the reagents needed for PGT-M library prep, including primer pool, Ampliseq reagents and barcodes.

Reagent t Kit

2

Includes access to the analysis software for free, with support from one of our FAS.

Software re An Analysi ysis

3

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.

For PGT-M

4

Combinable with Reproseq in one single workflow.

With PGT-A

Lab Protocol

PGT-A

Ion ReproSeq roSeq 4 h

Template

IA IA

Analysis

Ion Reporte rter

Sequencing

100 0 bp bp 1 h 2.5 h 2 h

PGT-M

PGD-Seq 4 h 3 ul

A/T T/T A/T

A T T T A T

SNP Phasing

A T T T A T

A/T A/T T/T A/T A/T T/T T/T

A T T T T T A T

SNP Phasing

A C T G T C T C A C T C

C/G C/C C/C C/C C/C G/C G/C

A T T T T T T A C C G C G C C C

SNP Phasing

A C G T G G T C T T C G A C G T C G

G/G G/G T/G G/G G/T G/G G/T

A T T T T T T A C C G C G C C C G T G G G T G G

SNP Phasing

A C G C T G G C T C T C T C G A A C G C T C G A

C/C C/A C/A C/A C/C C/A C/C

A T T T T T T A C C G C G C C C G T G G G T G G C C C A C C C A

SNP Phasing

A C G C T G G C T C T C T C G A A C G C T C G A A T T T T T T A C C G C G C C C G T G G G T G G C C C A C C C A

SNP Phasing

A C G C T G G C T C T C T C G A A C G C T C G A A T T T T T T A C C G C G C C C G T G G G T G G C C C A C C C A Carrier Carrier Normal Affected

SNP Phasing

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)

Non carrier 1st

st Cycle

cle (8 8 embryo ryos) s) 5 6 Euploid 3 Healthy embryos 2nd

nd Cycle

cle (10 0 embr bryo yos) s) Non carrier 5 7 Euploid 4 Healthy embryos

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

Normal Balanced

How it is Used

The kit is ordered according to PGT-M case (gene) and delivered.

Orderin ering

Sequencing and analysisof familial samples is first performed for PGT-M setup.

PGT-M Set up

IVF cycle begins and resulting embryos are eventually biopsied.

IVF Cycle

Biopsies are amplified and sequenced.

Amplifi ificat catio ion

Embryo results are analyzed via software and validated for final report.

An Analysis

PGD-Seq Features

Based d on NGS

The most efficient technology for DNA analysis

Extensive sively ly Validated dated

Validated in clinical use with 150 different monogenic disorders

Simplifi fied workflo flow

Simplifies the laboratory routine even allowing fresh transfers

Based d on SNPs

SNP linkage provides more robust analysis to avoid allele drop-out

Flexible le

Easy to customize to any need

Support t Center ter

Our support center assists from case preparation to data analysis

Platform Comparison

Ion ReproSeq roSeq PGD-Seq Karyo ryomapping VeriSeq

Aneuploidy uploidy screening ng Detecti tion

• n of poliploidy

ploidy Detecti tion

• n of segmenta

ntal aberrat ration

• ns

Detecion

• n of mitotic
• tic error
• rs (mos
• sai

aicism) Indire rect t Test Direct t Test (poi

• int

t muta tati tion

• n)

Fresh transfer Scala alable ble Price

Alain Rico Melanie Haessler

Miguel Fernández Rubén Dasí Helena Blanca Santiago González Santiago González-Reig

Acknowledgments

Santiago González-Reig Vanessa Penacho Marta Alfonso Diego Amorós Andrés Antón Helena Blanca Cristina Botella Tatiana Garijo Francisco Galán Alba López Irene Manchón-Trives Paula Brígido

Thank you!

luis.alcaraz@bioarray.es http://www.bioarray.es