Lecture 9b: Emerging technologies Flashcards
Applications of next-generation sequencing
table on slide 3
Next gen sequencing… an anlogy
analogy of a book:
* You’re told to look at a specific page
OR
* You can look at 5 pages………..(!)
– Random?
– Is there other information to suggest looking at certain pages?
- How do you know what’s correct and what isnt
– What separates Caitlin from Caitlyn?
– What happens when you really start looking hard and find there are many errors, but most of them arent critical? - What if you could screen al the important parts of EVERY page
quickly and economically?
– How do you identify what’s important from what’s not?
Exome Sequencing process…5
- dna sequence with:
Exon1..exon2..exon3…exon4….exon5 - FRAGMENT AND HYBRIDISE TO ‘NIMBLEGEN’ CAPTURE ARRAY
- elute
- 454 sequencing
- Analyse exon sequences
Example – Exome Capture
image on slide 7
Example – Exome Capture
Variant in NEB
image on slide 8
what is and what is the process of EXOME SEQUENCING? = 5
1 * A single method to sequence >250 000 exons
2 – Solution-phase oligonucleotide hybridisation
3 – ~35 000 exons not sequenced (mostly 5’UTRs, but also others)
4 – 45.1Mb of DNA sequenced per individual
5 – 1-2 weeks turnaround, $500-1000 per sample
Data Processing = 11
- Mapping/Variant calling (SNV and indel)
- Gene-lists
- NS exonic (inc frameshifts)
and splice site variants - Evolutionary conservation
- EXAC/GnomAD filter
- Other local filters/databases
- Inheritance patterns
- Other mapping input
- Genetic Pathologist input
- Confirmation in patient
- Confirmation in family members
Bioinformatics/Analysis:
- Alignment and variant calling
…. * Low stringency variant calling – attempt to minimise false negatives - 50000 variants
Bioinformatics/Analysis….
Positive Selection
Negative selection w/ Pathway genes
Screen specific genes Negative selection
Positive Selection
* Which database?
* ClinVar?
* HGMD?
* Database quality
variable
* Some entries incorrect * Exomes : 20-40
Screen specific genes
* Which genes?
* Which variants?
* Missense vs
synonymous
* 2-20 variants/gene
* Not all real
Negative selection
* Low freq in GnomAD
* Local databases
* Exonic, splicesites
* Non-synonymous only
* Exomes : 400-500 vars
Negative selection w/ Pathway genes
- Best combination yet
* Local databases
* Exonic, splicesites
* Non-synonymous only
* Exomes : 40-50 vars
Application of WES to Fetal Akinesia = 3
(Ravenscroft et al, submitted to Neuromuscular Disorders)
- Rare group of disorders with clinical/phenotypic heterogeneity
- Complicated by the fact that it occurs ‘in utero’
- Genetically heterogeneous
Application of WES to Fetal Akinesia =
Rare group of disorders with clinical/phenotypic heterogeneity = 5
– Decreased movement
– Intrauterine growth restriction
– Craniofacial anomalies
– Joint contractures
– Overlaps with lethal congenital contracture syndromes and multiple pterygium syndromes
Application of WES to Fetal Akinesia - Complicated by the fact that it occurs in utero = 3
– Makes diagnosis difficult – ultrasound 12 wks, 18wks
– Prenatal genetic testing possible only if the cause can be identified
– Likely only option is termination
Application of WES to Fetal Akinesia = Genetically heterogeneous
– Autosomal dominant, recessive and X-linked forms
– Likely to be many different genes involved
Case report
- Caucasian family, 2 pregnancies terminated with fetal akinesia and arthrogryposis = 5
– Absence of proximal musculature, thin diapraghm, pulmonary hyoplasia
– Dystropic muscle replaced by fat, with inflammation
– Absence of muscle proteins (IHC)
– Twin pregnancy diagnosed as Multiple Pterygium Syndrome (MPS)
– Sanger Seq of known MPS genes excluded mutations
* CHRNA1, CHRNG, DOK7
Caucasian family, 2 pregnancies terminated with fetal akinesia and arthrogryposis
DIAGRAM SLIDE 13
DIAGRAM OF CASE STUDY
SLIDE 14
CASE STUDY RESULTS…12
1 * Variants found in Glycogen-Branching Enzyme 1 (GBE1)
– Known essential splice-site mutation in both
– Novel missense mutation in Exon 7 (His319Arg) in both
– Father carried splice mutation, Mother carried missense mutation
2 * Muscle pathology re-examined
- Inclusions consistent with GSD IV (Andersen disease; OMIM 232500)
3 * Subsequent pregnancy offered prenatal testing for these variants
– 12 weeks scan suggested the same problem
– Sanger sequencing confirmed both mutations present
4 * Ideally, functional testing should be performed to confirm mechanism– Glycogen Branching Enzyme activity in case fibroblasts showed residual (6%)
activity (13U/g) compared to controls (200U/g)
– Glycogen debranching Enzyme activity similar to cases and controls
– Splicing mutation suggested to result in some residual function
WES of Families with Charcot-Marie-Tooth Disease (CMT) = 9
INHERITANCE?
SYMPTOMS?
SUBTYPES?
CLINCIAL AND MOLECULAR
1 * Common inherited peripheral neuropathy
…2 – mutations in 54 known genes.
3 * Distal wasting of the legs and later hands.
…4 – Skeletal deformations including pes cavus and hammer toes.
5 * 3 subtypes:
…6 – CMT1 : demyelinating
…7– CMT2 : axonal
…8 – intermediate CMT : mixed
- The clinical and molecular heterogeneity of this disease means many families remain without a molecular diagnosis
– >400 in Western Australia
- The clinical and molecular heterogeneity of this disease means many families remain without a molecular diagnosis
CMT pedigree
SLIDE 17
Linkage and CNV analysis FOR CMT = 3
1 * Linkage analysis excluded all known CMT genes except a 65Mbp region on chromosome 16 including three known CMT-causing genes.
2 * AARS (alanyl-tRNA synthetase) fitted with the autosomal dominant inheritance inthe family.
3 * CNV of all known CMT regions were excluded
- DIAGRAM ON SLIDE 18
AD = Autosomal Dominant
RI = Recessive Intermediate
AR = Autosomal Recessive
Exome-sequencing Results
TABLE ON SLIDE 19
a
represents the algorithm applied determined by the mode of inheritance.
b
represents the list of variants that are observed at least 8X in 16 WES runs.
TABLE ON SLIDE 20
AARS:uc002eyn.1:exon8:c.G986A:p.R329H, chr16 70302259 70302259 C T 1 111
Confirmation by Sanger sequencing…4
- CONTROL
- PATIENT
- cDNA
- AA sequence
diagram on slide 21
pedigree + sangers technique
slide 22
After 30 years of stability, sequencing is now a rapidly
evolving technology = 3
– Cost decreasing faster than Moore’s law
– Lab techniques are evolving all the time
– Moving more towards total automation
How soon until routine WGS is common? 4
– $1000? Less?
– Types of sequencers needed?
– Where are the bottlenecks?
– Where are the jobs?
