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 promotingany use or application of Thermo Fisher Scientific products presented by third parties duringthis seminar. Information and materials presented or provided by third parties are providedas-is and without warranty of any kind, including regarding intellectual property rights andreported results. Parties presenting images, text and material represent they have the rightsto do so.

Speaker was provided travel and hotel support by Thermo Fisher Scientific for thispresentation, but no remuneration.

Simplified Workflow

Easy to combine in one single protocol.

1 PGT-A + M

Less sample handling.

2 Risk reduction

Less investment in equipment and human resources.

3 Cost reduction

Any patient can opt.

4 Affordable

PGT-SR

IndirectTest

STRs

IsStructural?

D-5Biopsy

D-3Biopsy

aCGHaCGHNGS

Normal Abnormal

Normal

Abnormal

IsBig? HR-CGH

Normal AbnormalBIOPSY

Is PointMutation?

PGT-A

Yes

PGT-MNo

DirectTest

miniSeqYes

No

IndirectTest

STRs

IsStructural?

Normal

Abnormal

REPROSEQ

Normal Abnormal

BIOPSY

Is PointMutation?

PGT-MNo

DirectTest

miniSeqYes

No

Direct and Indirect Test

Direct Test Indirect Test

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?

1Includes all the reagents needed for PGT-M library prep, including primer pool, Ampliseq reagents and barcodes.

Reagent Kit

2Includes access to the analysis software for free, with support from one of our FAS.

Software Analysis

3For 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

4Combinable with Reproseq in one single workflow.

With PGT-A

Lab Protocol

PGT-A

IonReproSeq

4 h

Template

IA

Analysis

IonReporter

Sequencing

100 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/TA T T T T TA T

SNP Phasing

A

C

T

GT

C

T

C

A

C

T

C

C/G

C/C

C/C

C/C C/C G/C G/CA T T T T TTA

C C G C G CC 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/TA T T T T TTA

C C G C G CCC

G T G G G TGG

SNP Phasing

A

C

GC

T

G

GC

T

C

TC

T

C

GA

A

C

GC

T

C

GA

C/C

C/A

C/A

C/A C/C C/A C/CA T T T T TTA

C C G C G CCC

G T G G G TGGC C C A C CC A

SNP Phasing

A

C

GC

T

G

GC

T

C

TC

T

C

GA

A

C

GC

T

C

GA

A T T T T TTA

C C G C G CCC

G T G G G TGGC C C A C CC A

SNP Phasing

A

C

GC

T

G

GC

T

C

TC

T

C

GA

A

C

GC

T

C

GA

A T T T T TTA

C C G C G CCC

G T G G G TGGC C C A C CC A

Carrier Carrier NormalAffected

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.1597delA (p.Thr533LeufsX25)

Non carrier

1st Cycle (8 embryos)

5

6

Euploid

3 Healthy embryos

2nd Cycle (10 embryos)

Non carrier

5

7

Euploid

4 Healthy embryos

PGD-Seq Validation

PKD1:c.7292T>A (p.Leu2431Gln)

Performance

PGD-Seq Validation

PKD1:c.7292T>A (p.Leu2431Gln)

PerformanceFull concordance

Balanced Translocations

Balanced NormalBalanced Normal

How it is Used

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

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.Amplification

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

PGD-Seq Features

Based on NGSThe most efficient technology for DNA analysis

Extensively ValidatedValidated in clinical use with 150 different monogenic disorders

Simplified workflowSimplifies the laboratory routine even allowing fresh transfers

Based on SNPsSNP linkage provides more robust analysis to avoid

allele drop-out

FlexibleEasy to customize to any need

Support CenterOur support center assists from case preparation

to data analysis

Platform Comparison

Ion ReproSeqPGD-Seq

Karyomapping VeriSeq

Aneuploidy screening

Detection of poliploidy

Detection of segmental aberrations

Detecion of mitotic errors (mosaicism)

Indirect Test

Direct Test (point mutation)

Fresh transfer

Scalable

Price

Alain RicoMelanie Haessler

Miguel Fernández

Rubén Dasí

Helena BlancaSantiago González

Santiago González-Reig

Acknowledgments

Santiago González-ReigVanessa PenachoMarta AlfonsoDiego AmorósAndrés AntónHelena BlancaCristina BotellaTatiana GarijoFrancisco GalánAlba LópezIrene Manchón-TrivesPaula Brígido

Thank you!

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