Exome
Exome
Exome
2- Guidelines / Recommendations
http://www.genome.gov/sequencingcosts/
Cost driven test ordering change
$ ≈ $
FBN1 Aortapathy (Marfan and
Sanger sequencing Marfan like syndromes)
10 gene NGS panel
Total Number of Cases in ARUP
Next Generation Sequencing Lab
60
50
40
30
Total number of cases
20
10
0
Jan-Mar 2012 Apr-Jun 2012 Jul-Sep 2012 Oct-Dec 2012 Jan-Mar 2013
What is Exome sequencing ?
The sequence of all exons of the genome
What is missing?
Some protein coding genes
Some exons of some genes
Non-genic control elements
Copy number changes
Structural changes
mtDNA
Some microRNA genes
Why Exome Sequencing?
Focuses on the part of the genome we understand best,
the exons of the genes
Intellectual disability
Patient specific:
- well defined findings
- good evidence for a genetic basis
Family specific:
- affected family members
- inheritance pattern
Analytic Considerations
Bioinformatic aspects
- variant calling
- filtering
- analyzing genes only in Human Genome
Mutation Database or OMIM
- analyzing genes on mandatory reporting
Postanalytic Considerations
Reporting
- negative, positive, uncertain for primary patient finding
Patient consent
Barcoding
Cluster generation 1 day
Library prep
Enrichment
Barcoding
Cluster generation
Sequencing
Data Analysis
CLINICAL EXOME SEQUENCING
Work flow :
DNA (Sheared DNA) DNA fragments
Library prep
Repair and prepare ends
Ligate adapters
Enrichment
Barcoding
Cluster generation
Data Analysis
CLINICAL EXOME SEQUENCING
Work flow :
DNA (Sheared DNA) λmax
157 bp
After Sonication
Library prep 150-200 bp desired
Enrichment
Barcoding 244 bp
Cluster generation After adapter
binding
Sequencing
Data Analysis
Peak shift indicates successful library generation
CLINICAL EXOME SEQUENCING
Work flow :
DNA (Sheared DNA)
Coverage
Enrichment
Barcoding
Cluster generation
Sequencing
Data Analysis
Biotinylated RNA library baits covers all exons annotated in the
consensus CDS database as well as flanking sequence for each
targeted region and small non-coding RNAs
Work flow : CLINICAL EXOME SEQUENCING
DNA (Sheared DNA)
Library prep
Enrichment
A flow cell attached Hybridization of enriched Bridging
Barcoding with adaptors DNA to flow cell
Cluster generation
Sequencing
Library prep
Enrichment
100bp
Barcoding
Cluster generation
Sequencing
Data Analysis
T A C G - - -
Work flow :
DNA (Sheared DNA)
Library prep
Enrichment
Barcoding
Cluster generation
T G C A
Sequencing
Enrichment
Barcoding
Reference sequence
Cluster generation
Library prep
Enrichment
Barcoding
Cluster generation
Sequencing
Data Analysis
Work flow :
DNA (Sheared DNA)
Library prep
Enrichment
Barcoding
Cluster generation
Sequencing
Data Analysis
GUIDELINES/REGULATIONS
CLIA/CAP/ACMG
Guide validation of samples, analysis and reporting
Method (exome Agilent SureSelect Agilent SureSelect, NimbleGen NimbleGen SeqCap Agilent SureSelect Agilent SureSelect
capture) NimbleGen SeqCap (custom
designed) VCRome
2.1
Variant + Only primary + Only primary + Primary, some + Primary, all + Only primary + Only primary
confirmation secondary results secondary results
Jamal SM, Yu J-H, Chong JX, Dent KM, Conta JH, Tabor HK, Bamshad MJ. 2013. Practices and policies of clinical exome sequencing providers: Analysis and implications. Am J Med Genet Part A 9999:1–16
Exome Interp Algorithm: weekly meeting
Variants (SNV)s in 20-25,000 genes, ~ 20K-30K
SNVs ~2,000
Bioinformaticist
Sanger Confirm/Report
Bioinformatics Pipeline: NGS
Variant Viewer
Brendan O’Fallon: Bioinformaticist at ARUP
Pop. frequency:
e.g. Exclude all var with pop
frequency greater than 0.01
Exon effect: e.g. Exclude
var intergenic, intragenic,
UTR
Quality & Depth
Deleterious Score: SIFT,
PolyPhen, Mutation Taster
Genes & Regions
42 total, 2: autovalidation, 7:
noonan, 2: marfan, 31: HHT
c. 1699G>A, p.V567I
c. 184G>A, p.V62M
c.4204G>T, p.E1402X
Case 3: ARID1B
ARID1B: At-rich interaction domain-containing
protein 1B
Santen et al, 2012, Nature
Genetics:
“de novo truncated mutations
in ARID1B gene in three
individuals with Coffin-Siris
syndrome”
Case 3: Coffin-Siris
Globe developmental delay
Short stature
Feeding difficulties
Hypotonia
Moderate to severe learning difficulties
Broad thumbs and toes
Posterior rotated ears