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General presentation of NGStechnologies: WES/WGS
9thPanhellenic Interdisciplinary Conference onAlzheimer's Disease & Related Disorders
&1st Mediterranean on Neurodegenerative Diseases
Vogiatzi Emmanouella
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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
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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
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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
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Family vs sporadic cases for WES/WGS analysis
Size
eff
ect
Population frequency
Family studies
Sporadic studies
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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)
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Types of mutations
Gene andmutations
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WESprocedure
Biesecker, NEngl J Med,2014
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next step: variants annotation to obtain information such as genomic position functional effect (eg, missense, nonsense variants)
final step: identification of causal mutations
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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
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Ku et al, Ann.Neurol, 2012
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WESvs
WGS
Biesecker, NEngl J Med,2014
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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
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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
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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
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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
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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
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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
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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