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
