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Preimplantation Genetic Testing for Monogenic/Single Gene Disoders (PGT-M, PGD) Luis A. Alcaraz
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
