DON LOVE DIAGNOSTIC GENETICS

FISHKARYOTYPING

SEQUENCING

PROTEIN MODELLING

QUANTITATION

CNV

GROWTH DEMANDS REDESIGN:A CASE STUDY OF MOLECULAR GENETICS

GENES IMPLICATED IN LONG QT SYNDROMEGENES IMPLICATED IN LONG QT SYNDROMEClassification Gene Chromosome Ion Channel Type Frequency Types of Mutation

LQT1 KCNQ1

KCNH2

SCN5A

ANK2

KCNE1

KCNE2

KCNJ2

CACNA1c

Cav3

SCN4B

LQT2

Mainly missense, insertion, deletions

30-50%IKs α-subunit11

7

3

4

21

21

LQT3

2

Mainly missense and insertions, deletions

12

25-45%

3

IKr α-subunit

Na+ α-subunit

Adaptor protein

IKs β-subunit

LQT4

IKr β-subunit

K+ channel

Mainly missense and deletions

Ca2+ channel

Membrane domain

5-10%

<1

<1

<1

LQT5

<1

<1

11

Missense

Missense and deletions

Missense

Missense and deletions

Na+ β4-subunit

<1

LQT6

LQT7

<1

Missense

Missense

Missense

LQT8

LQT9

LQT10

A SIMPLE GENE COMPRISING TWO EXONS

E1 E25’3’

5’ 3’

PRINCIPLES OF PCR AMPLIFICATION

3’ 5’

5’ 3’

5’ 3’

3’ 5’

5’ 3’

5’ 3’

5’ 3’

3’ 5’

3’ 5’

FIRST ROUND

SECOND ROUND

Exponential increase of the number of copies during PCR

PCR AMPLIFICATION

M13-FOR TGTAAAACGACGGCCAGTM13-R EV CAGGAAACAGCTATGACC

SIMPLICITY OF PRIMER DESIGN

E1 E2

Annealing TmMgCl2

GC Buffer

58°C1.5mM

+

60°C, 2.5mM

EVOLUTION OF CHAOS

ONE GENE MANY GENES

Five annealing temperaturesFour Mg concentrationsEach amplicon sequenced using two gene-specific oligos

56°C, 2mM 57.5°C, 1.5mM

55°C, 1mM 62°C, 1mM

Tm 60°C2.0mM MgCl2+/-GC Buffer

Fast Start Taq DNA Polymerase

M13 tails for all primers

AIM: TO ACHIEVE SIMPLICITY IN PRIMER DESIGN AND AMPLIFICATION

EXON-SPECIFIC PRIMER PAIR

chr16:67406743+67407333 591bp TTGAGAAGCCATGGTAAGTAATTG GAAGGGAACAGGTGAAAGGAGTTGAGAAGCCATGGTAAGTAATTGtggtttctgccattgaaagtcatggcagaaaccacagttacttttgcaccaacctaatatattaccaaaagcaacagttaaggatttaattttatttttactaacacaaaatgtttcgttttgtttttaacttcattgtttctgctctctagggcttggattttgaggccaagcagcagtacattctacacgtagcagtgacgaatgtggtaccttttgaggtctctctcaccacctccacagccaccgtcaccgtggatgtgctggatgtgaatgaagcccccatctttgtgcctcctgaaaagagagtggaagtgtccgaggactttggcgtgggccaggaaatcacatcctacactgcccaggagccagacacatttatggaacagaaaataacgtaagtgtgaggatttttcaactgacttgcagcaactggttattttatatcattttatatgtaaatcaataatatgtacttcatggcattttgtcatttgtctgtacaagaccattctcttaatttattttttattccctttatctgtgCTCCTTTCACCTGTTCCCTTC

Forward: 59.6 C ttgagaagccatggtaagtaattgReverse: 60.1 C gaagggaacaggtgaaaggagThe temperature calculations are done assuming 50 mM salt and 50 nM annealing oligoconcentration.

EXON20bp 20bp

Splice acceptor site Splice donor site

AMPLICONS: LQT GENE EXONS IN PLATE FORMAT

CODING SEQUENCE OF LQT GENES 1, 2, 3, 5, 6 AND 7

96 well384 well

CURRENTLY USE CAPILLARY-BASED SEQUENCING

LIQUID HANDLING ROBOT

AMPLICON PURIFICATION (Exosap; Ampure)

SEQUENCE SET-UP

SEQUENCE CLEAN-UP (Cleanseq)

LABORATORY AUTOMATION

SEQUENCE ANALYSIS SOFTWARE

SeqMan (DNASTAR)Sequencher™ (GENE CODES CORPORATION)Mutation Surveyor®/Explorer® (SOFTGENETICS)StadenSeqScape® (APPLIED BIOSYSTEMS)

Variant Reporter™ (APPLIED BIOSYSTEMS)

IMPORT GENBANK SEQUENCE WITH ANNOTATIONSor

IMPORT YOUR OWN REFERENCE SEQUENCE AND ANNOTATE MANUALLY

GENE CONNECTION FOR THE HEARThttp://www.fsm.it/cardmoc/

THE FUTURE IS NOW

THINK DIFFERENTLY.

JUST DO IT!

NEXT GENERATION: PARALLEL (DEEP) SEQUENCING

PCR AMPLIFY REGIONS OF INTEREST

POOL PRODUCTS

SEQUENCE(BY SYNTHESIS)

HIGH THROUGHPUT PARALLEL SEQUENCING:ROCHE GS-FLX SEQUENCING PLATFORM

HIGH THROUGHPUT PARALLEL SEQUENCINGROCHE GS-FLX SEQUENCING PLATFORM

ABILITY TO POOL PATIENTS’ AMPLICONS

REQUIRES DIFFERENTIALLY TAGGED(BAR CODED)

PRIMERS FOR EACH PATIENT

HIGHER LEVEL POOLING CONCEPT

PARALLEL SEQUENCING APPROACH

PCR AMPLIFY REGIONS OF INTEREST

POOL PRODUCTS

SEQUENCE

FRAGMENT GENOMIC DNA

ADD LINKERS

CAPTURE DEFINED AMPLICONS

ELUTE CAPTURED AMPLICONS

SEQUENCE

AGILENT SURE-SELECTARRAY-BASED CAPTURE

GS-FLX SEQUENCING

Albert et al, Nature Methods 2007;4:903-905

ARRAY-BASED CAPTURE

GS-FLX SEQUENCING

DON LOVE DIAGNOSTIC GENETICS

FISHKARYOTYPING

SEQUENCING

PROTEIN MODELLING

QUANTITATION

CNV

MODERN TRENDS IN GENOME ANALYSIS:CAN WE DO IT, SHOULD WE DO IT?

1

2

ARRAY CGH

AFFYMETRIX SNP 6.0 CHIPS

CHARACTERISATION OF CHROMOSOME 10 INTERSTITIAL DELETION

ARRAY CGH : 46,XX,del(10)(q23.1q23.2)

ALTERNATIVE ANALYSIS OF SNP DATA USING RECENTLY RELEASED AFFYMETRIX SOFTWARE

ARRAY CGH : 46,XX,del(10)(q23.1q23.2)

IDENTIFICATION OF GENES THAT

LIE IN THE DELETED REGION

The deletion resolved to 81,619,836bp-89,084,996bp on chromosome 10Multiple genes are located within this region

Susceptibility to severe respiratory syncytial virus infection

Brain demyelination through MAT1A deficiency

Susceptibility to schizophrenia

Predisposition to juvenile polyposis

ARRAY CGH : 46,XX,del(10)(q23.1q23.2)

ARRAY CGH: NEW WORK FLOWGENOMIC DNA

WHOLE GENOME AMPLIFICATION100ng, 30ºC, 3 HOURS

ENZYMATIC CLEAVAGEAND LABELLING OF FRAGMENTS

HYBRIDIZATION TO CHIPWASH CHIPSCAN CHIP

THURSDAY

FRIDAY

THE FOCUS IS ON TRISOMY 21

NON-INVASIVE PRENATAL DIAGNOSIS (NIPD)

SEQUENOM CORE TECHNOLOGY IS BASED ON SEQUENOM CORE TECHNOLOGY IS BASED ON MALDIMALDI--TOF MSTOF MS

1

+

+

+

+

• Samples spotted on chip• Chip is placed in machine• Machine is vacuum tube with electric field with

detector at end of tube

• Laser “zaps” sample• Laser tuned such that molecules get same

charge

• Molecules “zapped” by the laser become charged

• Samples accelerate in electrical field• Smaller molecules accelerate faster

• Molecules hit detector• Order of peak indicates size of molecule• Area of peak indicates abundance

1

12

13

14

15

+

+

+

6600 6700 6800 6900

UEP.M

MBT01167CC

T

CTT

UEP.M

MBT05641 CC

T

CTT

UEP.M

MBT05728

UEP.M

MBT10830

MMBT05728

Mass

A T G T

10 mer tag

10 mer tag

10 mer tag

10 mer tag

A TT A

G TC A

A G

Primer ExtensionA,C,T,G terminators

PCR

Sample ConditioningSpot on SpectroCHIPFly on Mass Spec

SAPHybridize Extension Primer

OVERVIEW OF GENOTYPING ASSAYOVERVIEW OF GENOTYPING ASSAY

Allele 1 Allele 2

A GA

normal fetus trisomy 21 fetus

SNP on PLAC4(chromosome 21)

transcription

PLAC4 RNAexpressed in

placenta

deviation in RNA-SNP

allelic ratio

release into maternal circulation

circulating PLAC4RNA in maternal

plasma

deviation in RNA-SNP

allelic ratio

GA

RNA-SNP ALLELIC RATIO

PCR amplification

Relative peak area: 0.5 0.5 0.67 0.33

Allelic ratio = allele-G peak areaallele-A peak area

1 0.5

Base extension

Mass detection

Mass

Inte

nsity

allele-Aallele-G

Mass

Inte

nsity

allele-Aallele-G

RNA cDNA

normal fetus trisomy 21 fetus

RNARNA--SNP ALLELIC RATIOSNP ALLELIC RATIO

CHALLENGESCHALLENGES

BIOLOGICAL RELEVANCECLINICAL UTILITY

INFRASTRUCTURE DEVELOPMENTIMPLEMENTATION

ACKNOWLEDGEMENTSElaine Doherty

Anne Vaughan, Debbie Prosser, Jenny LoveAnna Zhang, Stella Lai

Christina Lim, Jamie-Lee Day