General presentation of NGStechnologies: WES/WGS

9thPanhellenic Interdisciplinary Conference onAlzheimer's Disease & Related Disorders

&1st Mediterranean on Neurodegenerative Diseases

Vogiatzi Emmanouella

Next-Generation sequencing technologies(NGS)

Can be applied in:- small families- sporadic patients

Deliver novel insights with even smaller number of patients

Simultaneous screening of a whole battery of genesimplicated in disorders

personalisation of treatment for neurodegenerative disorders

Application of NGS technologiesin the identification of disease-relevant variants

Odds Ratio: the magnitude of a variant’s effect on disease risk

Majewski, J.Med.Genetics, 2011

Question: under what circumstances is the cosegregation of a variant withdisease expected to be greater in families than in unselected affectedindividuals? the use of familial cases selectively increases the frequency of the variants

with the greatest contribution to the disease

(Helbig, European Journal of Human Genetics, 2013)

Family-based approach

Cosegregation of rare variants withdisease

multiple affected individuals

Family vs sporadic cases for WES/WGS analysis

Size

eff

ect

Population frequency

Family studies

Sporadic studies

Whole Exome Sequencing (WES)

targeted sequencing of the protein-coding portion of thehuman genome

diagnostic approach for the identification of moleculardefects in patients with suspected genetic disorders

Variant Identification (disease-causing mutations)

Types of mutations

Gene andmutations

WESprocedure

Biesecker, NEngl J Med,2014

next step: variants annotation to obtain information such as genomic position functional effect (eg, missense, nonsense variants)

final step: identification of causal mutations

Whole exome/genome sequencing’s needle-in-a-haystack problem

• variants that truly matter to an illnessvs

• the great number of functional variants present in every genome

Guideline direction:approaches to identify causal mutations

Ku et al, Ann.Neurol, 2012

WESvs

WGS

Biesecker, NEngl J Med,2014

kits for exome capture and enrichment performed:

• Agilent• Nextera Rapid• Nimblegen

NGS technologies’ sequencers for WGS/WES:

• Illumina Genome Analyzer or HiSeq 2500• Life Technologies ION Torrent• Life Technologies SOLiD• Roche 454 Genome Sequencer• Pacific Biosciences

Coverage:times that each nucleotide of the whole genome (or exome) is read and aligned An average coverage of 30-fold is sufficient (accurate calling of variants)

human

WGS/WES possibilities

Kit Mb coverage price (persample)

bioinformaticanalysis TOTAL

Nextera Rapid CaptureExpanded Exome Kit 62 105X 1.085 € 125 € 1.210 €

Agilent AV5 Exome Kit 51 50X 755 € 111 € 866 €

Ion PI™ Hi-Q™OT2 200Kit 39,5 94% >10X 470 € 46 € 516 €

Roche NimbleGenSeqCap EZ V3 Kit 34 >20X 580 € 50 € 630 €

Nextera Rapid CaptureExome Kit 37 45X 615 € 31 € 646 €

megabases to gigabases of nucleotide sequence from asingle instrument run, depending on:

Platform X Coverage

0

2

4

6

8

10

12

14

16

18

20

Feb-14 Mar-14 Apr-14 May-14 Jun-14 Jul-14 Aug-14 Sep-14 Oct-14 Nov-14 Dec-14 Jan-15 Feb-15 Mar-15

patients

WES for diagnostic purposes

Feb-14 May-14 Aug-14 Nov-14 Dec-15 Feb-15 Mar-15patients 1 1 18 10 12 13 17

δείγματα που έχουναναλυθεί

δείγματα που είναι προςανάλυση

δείγματα που αναμένεται ναανοίξουν

Analysed samples 44Samples to be analysed 11Samples in wait for the procedure 17

TOTAL: 72

"θετικά" (πρωτεύονταευρήματα)

"αρνητικά" (δευτερεύονταευρήματα ή κανένα εύρημα)

Analysed samples 44“positive" (pathogenic variant) 25

“negative" (no findings) 19

Statistics

novel αλλαγές

αλλαγές που έχουναναφερθεί (biblio)

“positive" samples 25novel variants 1Variants in biblio 24

“positive" samples 25Sanger validation samples 11Samples in wait for Sangervalidation 14

Statistics

Example: variant annotation and identification of causal mutations

Variants data for 1 patient

Reference genes >19.000

Total identification of variants ~ 150.000

after exclusion of intronic andsynonymous variants

~ 20.000 missense/nonsensevariants

Probably pathogenic variants (afterexclusion of functional variants presentin every genome)

~ 140

after exclusion of common variants(present in a specific population, e.g.the Greeks)

~ 30-40

Pathogenic variants (causal mutations) 0-2

Validation through Sanger sequencing 0-2

Illumina Sequencing

http://www.youtube.com/watch?v=l99aKKHcxC4

Exome sequence and analysishttps://www.youtube.com/watch?v=5bNYJ_Kb8ko

Oxford Gene Technology: detecting rare disease variants

http://www.ogt.co.uk/genefficiency_rare_disease_family_trio_sequencing_service