Duncan - testing protocol, NGS and microarrays Flashcards
briefly describe the structure of genomic medicine services in the UK, including the sorts of things they test for
7 genomic hubs, each with a load of labs
Perform genomic testing in their areas and specialist testing for the population
test for:
Rare disease - germline disorders (i.e. variant/mutant present in every cell since birth)
Cancers - hereditary cancers (variant/mutant in every cell), and somatic cancers (e.g. leukaemia, genetic ‘aberration, is not in every cell only a proportion)
briefly, what is done with the results of genomic testing in the UK?
Results -
are then passed on to the appropriate consultants
The patient (hopefully) receives a diagnosis and can understand the cause of their problem - offers relief
Understand their prognosis (future implications) to influence lifestyle choices
Risk to family allows for decisions around testing others
Pharmacogenomics - the specific mutation can tell us if certain treatments won’t/will work sometimes
give an explanation of the difference between genomics and genetics.
include a little bit on where our knowledge is lacking
Genomics = you’ve sequenced the whole genome
Genetics = you’ve focused/sequenced a specific gene
We have 20,000 protein-coding genes, thats 1% of the genome
Disease-causing variation is in the 1%
We’re not really sure about the other 99%. We don’t really look into introns, microRNAs are something we know that turn genes on and off, but enough research has not been done/techniques developed to commonly use this
when deciding what kind of/amount of sequencing needs to be done, what are the options and when would you pick them?
Single gene sequencing -
If we have a strong suspicion of the gene involved, or a family member has a known mutation, e.g. Cystic Fibrosis (Sanger)
Targeted gene panels -
Next generation sequencing of a set of genes linked to a particular condition, e.g. colorectal cancer, heart disease, for when symptoms point to a certain condition
Exomes -
Next generation sequencing of the protein-coding only sequences in the genome, e.g. DDD Study - Deciphering Developmental Disorders Study (DDD) is to determine if new high-throughput genetic technologies can determine the cause of developmental disorders, and to facilitate the use of these technologies in the NHS
Genomes -
Next generation sequencing of the whole genome, e.g. 100K Project
These last two tend to give a diagnosis 20-30% of the time
in terms of technologies used in genetic testing, just name 7
- FISH
- Karyotype
- Oligonucleotide arrays
- SNP arrays
- MLPA
- Sanger sequencing
- Next Generation Sequencing
what are some drawbacks of Sanger sequencing?
Requires a set of primers for every exon, which is incredibly time consuming to prepare, and you can only do one gene at a time
explain the NGS ‘pipeline’ designed to reduce workload
100 gene NGS panel may give use 250 variants. This is too many to analyse.
So first we want to remove those variants that are common in the normal population. Done using human genome databases like gnomad database
In our example this could leave us with 20 variants.
We can then remove more that may be synonymous or located within non-coding DNA.
This should leave us with only 1 or 2 variants to analyse
what are some benefits/strengths of NGS?
NGS allows us to analyse 1 to 1000s of genes, even the whole genome.
Fast - results reported within 8 weeks of receiving a sample (In comparison, Sanger sequencing can only examine one gene for one patient at a time.)
Using DNA barcodes we can mix patient DNAs and run them in one assay (so one eppendorf, massively reduces
We can diagnose more diseases with one test
MPLA = Multiplex Ligation-dependent Probe Amplification
give an overview of the steps involved, and of its applications.
Steps in MLPA:
Probe Hybridization:
Synthetic probes bind to specific DNA sequences.
Probes hybridize adjacent to each other on the target sequence.
Ligation:
Ligase enzyme joins adjacent probes if they match perfectly.
Ensures high specificity.
Amplification:
Ligated probes are amplified using PCR with universal primers.
Each probe produces a uniquely sized product.
Detection:
Products are separated by capillary electrophoresis.
Relative peak heights indicate copy number changes
APPLICATIONS:
- Copy Number Variation (CNV) Detection:
Detects deletions/duplications in disease-related genes.
E.g., DMD gene mutations in Duchenne/Becker muscular dystrophy
- Cancer Testing:
analyses gene amplifications or deletions in tumour suppressors/oncogenes - variation known as MS-MLPA (methylation specific MLPA) can be used to detect methylation disorders like Prader-Willi Syndrome
briefly, how does karyotyping work?
Looking at the banding can allow you to detect changes around 5-20 MBs long, which is quite large.
Not used as much now, but still important under several circumstances
Process - Trypsin produces dark and light bands
Dark - AT rich, late replicating, gene poor, heterochromatic regions
Light - GC rich, early replicating, gene rich, euchromatic regions
Analysis Count and pair up chromosomes
Match up bands, look for missing or extra chromosomes / bands
briefly, what are oligonucleotide microarrays used for?
detects deletions/duplications by fluorescently labelling patient DNA, applying to an array of oligonucleotides, DNA hybridises to probes, compare fluorescence levels to controls
0 = normal, does not differ from control
> 0.5 = gain, e.g. duplication
< -0.5 = loss, e.g. deletion
what are the 8 steps involved in NGS (start to finish)?
- library preparation - fragment your DNA sample
- adding DNA adaptors containing your PCR primer binding site
3.
describe the first part of NGS, in which the patient sample is prepared, made to attach to the flow cell ready for amplification.
Fragment patient DNA sample into 200 base pair long chunks (note - these may overlap)
Add DNA adaptors to the fragments of patient DNA
these contain
1. a PCR primer binding site. All the adaptors have the same primer target site (so only one set of primers is required)
- a unique DNA barcode per patient(multiple patient samples at once, separate them out later)
- the adaptors also contain complementary sequences to the oligonucleotides that coat the flow cell. (two oligonucleotides all over the flow cell, each complementary to one of the two adaptors on the ends of the sample fragments)
There are two types of oligonucleotides, a forward and reverse sequence type (sense and anti-sense) allowing forward and reverse DNA sequences to attach to the flow cell.
The fragments get ligated to the flow cell via the oligonucleotide sequences complementary to those in the adaptors.
Once attached a polymerase will duplicate the attached fragment, creating a double stranded molecule…
once you’ve got your patient DNA fragments attached to the flow cell, what happens next in NGS? that is, how are the samples amplified?
Once attached a polymerase will duplicate the attached fragment, creating a double stranded molecule.
This double stranded DNA molecule is denatured (separated) and the template washed away
The fragments are then amplified via Bridge amplification.
For bridge amplification both ends of the fragment bind to the oligonucleotides on the flow cell via the adaptor sequences.
This forms a “bridge”.
Across this bridge polymerase duplicates the fragment creating a double stranded bridge.
The double stranded bridge is then denatured (separated) resulting in two complementary DNA fragments.
This process is then repeated many times.
Resulting in clusters of 100s of DNA fragments, each of which is derived from one original DNA fragment
in NGS, after bridge amplification, once you’ve got your clusters of DNA fragments, how do you go about sequencing them?
is it one fragment/cluster/patient at a time or…
process is called ‘sequence by synthesis.
Fluorescently labelled nucleotides (A, G, C and T, each emitting a unique fluorescence colour) are added to growing DNA strands, using the fragments in the clusters as templates.
As each nucleotide is added, laser excitation causes the characteristic fluorescence to be emitted.
A fluorescence detection scanner can then identify which nucleotide has been added to each strand.
A cluster could consist of 100s of DNA strands, hence one region of patient DNA is sequenced many many times
All DNA fragments are sequenced at the same time – parallel sequencing
