NGS application to neuroscience disease research ...€¦ · NGS application to neuroscience...
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Targeted next generation sequencing in inherited disease research NGS application to neuroscience disease research Alessandra Murgia Laboratory Molecular Genetics of Neurodevelopment University Hospital Padova Italy New products to enable discovery of de novo and germline mutations Sunday 28th May: 11:15–12:45 Room Ballerup
Transcript of NGS application to neuroscience disease research ...€¦ · NGS application to neuroscience...
Targeted next generation sequencing in inherited disease research
NGS application to neuroscience disease research
Alessandra Murgia Laboratory Molecular Genetics of Neurodevelopment
University Hospital Padova Italy
New products to enable discovery of de novo and germline mutations Sunday 28th May: 11:15–12:45
Room Ballerup
University Children’s Hospital Woman and Child Health Department
Padua, Italy
Molecular Genetics of Neurodevelopment
Laboratory Istituto di Ricerca Pediatrica (IRP CDS)
Who are we ?
Divisions of Child Neurology/Epilettology/Psychiatry
We study the genetic bases of neurodevelopmental conditions involving Intellectual disability, autism spectrum disorders, epileptic encephalopathies
and early onset epilepsy, sensorineural hearing loss
We are currently working with four NGS panels. A new cortical brain malformation panel is currently underway
NGS Volume of activity: 405 requests for panels 1/2016-5/2017
What do we do ?
The reasons of our choice
• NGS platform combining innovative sequencing system and technical features that respond to the needs of a single laboratory
• Involvement in specific disease areas • The possibility of a complete phenotypic characterization of the tested cases • The goal of studying the pathogenic role of disease-causing and «established»
candidate genes • The aim of generating immediately actionable data
We chose the Ion Torrent NGS Platform and opted for a targeted strategy
Next Generation Sequencing technologies
Neurodevelopmental disorders are clinically and etiologically highly heterogeneous and characterized by a wide range of overlapping clinical features
These characteristics pose serious difficulties for diagnoses based on
exclusive clinical criteria
Etiological information are highly needed to provide fundamental insights into the pathogenic mechanisms and pave the way to a fine understanding of the different phenotypes and the
variable clinical expression
How many possible genes involved ? How many disease-genes to be investigated ?
EOEE Targeted panel: the process of gene selection
>100 association studies with negative or not-reproducible results
Helbig and Abou Tayoun, Mol Syndromol 2016
Critical steps in the process of developing the “most appropriate tool”
Data mining
Gene prioritization : evaluation of a wide array of different evidences From gene expression to “omic” data
Specific biological and clinical knowledge Literature search
Phenotypic characterization
What if…we could use a large array of carefully prioritized genes
to investigate specific clinical topics ?
we could choose from this catalog
and develop a comprehensive multigenic panel
Ion AmpliSeq On-Demand Panels
Early Onset Epilepsy
Hearing loss
Intellectual disabilities
Autism Spectrum Disorders
ACTB COL11A1 EYA1 KMT2D NPC1 PRPS1 ARSA COL4A5 EZH2 LAMP2 NPC2 SCN9A ASPA COL9A1 FOLR1 MCOLN1 NPHP1 SMC1A
ATP1A3 CPT2 GALC MET OFD1 SMC3 ATP7A CSF1R GJA1 MID1 OTC SOX10 ATP7B DHCR7 GLI3 MLC1 PEX1 SPG7 BCOR DYNC1H1 HEXA MSRB3 PEX13 TJP2 CASR EP300 IDS MTHFR PEX26 TNC CBL ERCC6 KCNE1 MTOR PHF6 ZEB2
CEP290 ETFDH KDM6A NIPBL PLP1
59 genes panel
Disease Gene Catalog
Ion AmpliSeq On-Demand Panels: browse disease area
INTUITIVE AND FRIENDLY INTERFACE
• Standard gDNA • High specificity • Coding region (CDS only) • amplicons dimension: 125-275 bp
• Amplicons: 1828 • Pool1: 918 | Pool2: 910 amplicons • Panel size : 418,7 Kb
Disease Gene Catalog
Ion AmpliSeq On-Demand Panels: panel information
Ion AmpliSeq™ Library Kit Plus
Barcoding Xpress
Ion PGM™ Hi-Q™ View OT2 Kit
Ion PGM™ Hi-Q™ View Sequencing Kit
Chip 316v2 BC
Disease Gene Catalog
Ion AmpliSeq On-Demand Panels: kits and workflow
Software TMAP (Torrent Mapping Alignment): Signal processing, base calling, reads alignment with Torrent Suite software 5.2.2 Plug-in: Variant Caller Coverage analysis
Disease Gene Catalog
Ion AmpliSeq On-Demand Panels: data analysis
Experiment 1:
6 libraries 10 pM loaded on OT2 Hi-Q View
Experiment 2:
6 libraries + 4 libraries 20 pM loaded on OT2 Hi-Q View
Experiment 3:
10 libraries 10 pM loaded on OT2 Hi-Q View
Ion AmpliSeq On-Demand Panels: rationale
Mean Median Mode
Average
76 EOEE
experiments
177 196 277,7
Custom AmpliSeq Designer EOEE panel v3.6
316v2 BC:
2-3 million reads per run 300 Mb–1.0 Gb of sequence data
Experiment 1: 6 libraries, 10 pM loaded on OT2 Hi-Q View
Experiment 2: 6 libraries + 4 libraries, 20 pM loaded on OT2 Hi-Q View
Experiment 3: 10 libraries, 10 pM loaded on OT2 Hi-Q View
Ion AmpliSeq On-Demand Panels: report summary
Ion AmpliSeq On-Demand Panels: coverage analysis I
Panel size: 418,78 kb
Number of amplicons: 1878 mean max value min value
# mapped reads: 340280 1021960 87118
tot assigned amplicon reads: 322340 965082 83351
% on target: 94,9 96,9 92,7
average reads per amplicon X 176,3 527,9 45,6
uniformity of amplicon coverage % 98,7 99,2 98,16
amplicons with at least 20 reads % 98,6 99,78 91,3
amplicons with at least 100 reads % 70,9 98,36 2,9
amplicon with no strand bias % 97,3 99,12 90,8
amplicon reading e2e % 96,3 96,72 95,73
average base coverage depth X 186,1 552,1 48,02
uniformity of base coverage % 98,6 99,03 97,92
target base with at least 20 reads % 98,6 99,62 91,25
target base with at least 100 reads % 71,8 98,41 4,58
target bases with no strand bias % 96,9 98,45 90,98
e2e reads % 93,0 96,34 92,37
Experiment 1: 6 libraries, 10 pM loaded on OT2 Hi-Q View
Experiment 2: 6 libraries + 4 libraries, 20 pM loaded on OT2 Hi-Q View
Experiment 3: 10 libraries, 10 pM loaded on OT2 Hi-Q View
10 (0,5%) most recurrent AMPLICONS with total reads < 20X
mean max value min value
%GC 54.48 77.3 27,7
es: exon1, partial exon, exon-intron bundaries with poly T/A stretch…
Ion AmpliSeq On-Demand Panels: coverage analysis II
Experiment 1: 6 libraries, 10 pM loaded on OT2 Hi-Q View
Experiment 2: 6 libraries + 4 libraries, 20 pM loaded on OT2 Hi-Q View
Experiment 3: 10 libraries, 10 pM loaded on OT2 Hi-Q View
Mean Average variants /
libraries
238,8
Total variant: 770
Private variants: 205
Private variants, DP < 20X : 33 Private variants, DP ≥ 20X : 172
synonymous SNV 32
nonsynonymous SNV 46
frameshift deletion 0
frameshift insertion 1
nonframeshift insertion 0
nonframeshift substitution 1
nonframeshift deletion 1
synonymous SNV 6
nonsynonymous SNV 5
frameshift deletion 0
frameshift insertion 0
nonframeshift insertion 0
nonframeshift substitution 1
nonframeshift deletion 0
Ion AmpliSeq On-Demand Panels: variant caller
Experiment 1: 6 libraries, 10 pM loaded on OT2 Hi-Q View
Experiment 2: 6 libraries + 4 libraries, 20 pM loaded on OT2 Hi-Q View
Experiment 3: 10 libraries, 10 pM loaded on OT2 Hi-Q View
•Custom AmpliSeq Designer EOEE panel v3.6
•AmpliSeq Library kit 2.0
•Custom AmpliSeq Designer EOEE panel v3.6
•AmpliSeq Library kit Plus
Experiment 4: 10 libraries, 10 pM loaded on OT2 Hi-Q View
"On-Demand" genes or Library kit Plus ???
AmpliSeq Library kit Plus
AmpliSeq Library 2.0 kit (76 EOEE experiments) ∆
# mapped reads 322052,6 303521,5 18531,1 tot assigned amplicon reads 317523,8 278013,8 39510,0 % on target 98,6 96,9 1,7
average reads per amplicon 410,8 346,1 64,7 uniformity of amplicon coverage % 95,4 90,7 4,7 amplicons with at least 20 reads % 98,1 95,3 2,8 amplicons with at least 100 reads % 89,9 82,7 7,2 amplicon with no strand bias % 97,7 92,6 5,1 amplicon reading e2e % 94,7 73,9 20,4
average base coverage depth 422,3 316,0 106,2 uniformity of base coverage % 95,5 91,7 3,8 target base with at least 20 reads % 98,1 95,9 2,3 target base with at least 100 reads % 90,1 79,4 10,7 target bases with no strand bias % 96,6 84,2 12,4 e2e reads % 92,2 77,7 14,5
"On-Demand" genes or Library kit Plus ???
Greatly improved interface, user-friendly and readily available display of information
Large and growing curated gene inventory recognized as pathogenic and involved in major human genetic conditions
Allows viewing gene maps and expected coverage
Genes organized in different areas of pathology. Based on public databases this
provides essential information about each gene and associated phenotype/s and classify them hierarchically into categories
The repository genes can be arranged in any combination, to be analyzed in panels
of a wide range of sizes
Provides high flexibility, both in terms of content and possible integration, and the possibility of building «comprehensive» targeted panels
Wet analysis procedures are optimized
Ion AmpliSeq On-Demand Panels : in our view
Gene prioritization: work already done !
Fully informative interface
Flexibility of gene assembly into
panels of different composition and size
Optimal library chemistry Ion AmpliSeq Library Kit Plus !!!!!!
Key elements:
Molecular Genetics of Neurodevelopment Laboratory
Maria Cristina Aspromonte
Federica Cesca Emanuela Leonardi
Benedetta Scarpari
Roberta Polli Elisa Bettella
Thermo Fisher Scientific and its affiliates are not endorsing, recommending, or promoting any use or application of Thermo Fisher Scientific products presented by third parties during this seminar. Information and materials presented or provided by third parties are provided as-is and without warranty of any kind, including regarding intellectual property rights and reported results. Parties presenting images, text and material represent they have the rights to do so.
Ion Torrent and Ion AmpliSeq products referenced are For Research Use Only. Not for use in diagnostic procedures.
