Laboratory techniques Flashcards
What different techniques can be used to detect known mutations?
Allele specific PCR e.g. ARMs PCR for CF
restriction enzyme digest- when a mutation created or removes a restriction site e.g. F8 intron 22 inversion in maemophilia A
FRET hybridisation- e.g. for JAK2 V617F mutation detection in myeloid disease
Droplet PCR e.g. for BRAF V600E and EGFFR mutations testing in cancer (high sensitivity)
What are historic techniques for known mutation detection?
OLA PCR
Mini sequencing
Pyrosequencing
What are the benefits and limitations of known mutation detection
Benefits:
- quick, sensitive, simple.
- can be highly selective to target
Limitations:
-cannot detect novel mutations
Describe the basis of allele specific PCR
Allele specific PCR is a method for efficiently identifying specific SNvs
An allele specific forward primer (with different lenght stuffer sequences) and a common reverse primer amplify allele specific products which can be resolved by electrophoresis (usually capilliary).
Based on the observation that under suitable conditions a primer cannot extend with a 3’ mismatch. therefore only the primer that matches the allele(s) present will be amplified.
Give an example of allele specific PCR
the CFeu2 kit is based on ARMs PCR it is multiplexed to be able to test for the 50 most common mutations in the EU Caucasian population. Mutation differs for other populations and this should be incorporated in risk calculations.
- highly multiplexed assay. Uses A (mutant primers) and B (WT primers) tubes and also has primers to detect the polyT and TG tracts.
- as well as the 50 targeted mutations, indels can cause shifts in the B tube peaks. If detected this can indicate which exons should be sequenced to ID the causative variant
- products are distinguished by different fluorescent dyes and product lengths.
- products are separated by capillary electrophoresis and viewed using genotyping software e.g. genemarker
if a 3’ mismatch is werak a secondary mismatch can also be included to prevent non-specific amplification.
What are the benefits and limitations of Arms PCR?
Benefits
- quick, easy, simple
- does not require specialist equipment
- can be highthoughput
Limitations
- sensitivity of mutation detection is dependent on the population tested
- PSPs can result in false results- may result in a failure to amplify the mutant allele
- multiplexing can be difficult so commercial kits are often used
- sensitive ti MCC compared to methods of MCC detection (QF-PCR can exclude significant MCC to ~10%)
- need to confirm apparently homozygous results as it may in fact be due to a PSP or deletion.
- need to confirm compound het muts are in trans for CF
- if inheritance of a homozygous result can;t be confirmed consider: non-pat, deletion on other allele, PSP, UPD7, sample mix-up
Describe the use of restriction enzyme digest for known mutation testing
Restriction enzymes digest the DNA forming DSBs at specific sites known as recognition sites
- can be used when a mutation created or abolishes a specific restriction site.
- for diagnostic testing the mutation should create a restrictions site
1) sequence containing potential restriction site amplified by PCR
2) digested by restriction enzymes
3) products resolved by electrophoresis (capillary electrophoresis used most commonly in diagnostic labs)
it is good practice to include a second ‘control’ restriction site that will be digested in mutant and WT alleles to act as a digestion control.
can also be used for the detection of changes in methylation using enzymes that will only cut methylated DNA
Give an example of restriction enzyme digests used in diagnostic testing
- genotyping for the hemachromatosis C282Y variantby RSa1
- F8 intron 22 inversion in hemophilia A by bcII
what are the benefits and limitations of restriction enzyme digests?
- cheap, simple, quick, no specialist equipment required
- can only be used diagnostically if the sequence change created a restriction site. If the change abolishes a restriction site the exact sequence can’t be determined as it can be any base but WT
- polymorphisms can alter the restriction enzyme target sequence
- only suitable if a restriction enzyme is available for the target sequence of interest
- partial or overdigestion can affect interpretation
- not quantitative as hetero-duplexes formed in a heterozygous sample will not cut
What is FRET hybridisation?
Flourescence resonance energy transfer hybridisation
Uses flourogenic minor groove binders (MGB) whihc bind stably to ss DNA. These are bound to the 3’ end of a DNA probe and a flourescent quencher is bound to the other end.
MGB probes are designed to be specific to be specific for a single base, hence are susefl for allele discrimination assays.
Used for realt time PCR.
1) unbound probe free in solution, quenched by attached quencher
2) probe and primer binds to target DNA. probe remain3 quenched
3) DNA pol extends and from the primer. When it reaches the probe the 5’-3’ exonuclease activity degrades the probe, releasing the the flourescent reported from the quencher
4) the florescence emitted in measured in real-time e.g. Roche light cycler
Give an example of FRET hybridisation used diagnostically
Used for JAK2 V617F mutation testing in Myeloid disease (MF, PMF, ET)
Describe droplet digital PCR
methods fractionates sample into 1000;s of partitions (e.g. oil droplets in water-emulsion) so that each contains a single copy of the template DNA
PCR is run in 1000’s of parallel reactions using WT and mutant taqman probes (flourescent probe and quencher) and standard primers for region of interest.
software reads +ve and negative reactions and can distinguish Wt from mutant
Give an example of ddPCR in diagnostic use
BRAF V600E and EGFR mutation testing in cancers
What are the benefits and limitations of ddPCR?
Benefits:
- provides absolute quantification (assuming molecula distribution follows poisson distribution)
- can detect low levels of mutant against a hgh level of background so ideal for detecting mosaicism of mutants in cancer samples.
- in RT-PCR amount of target molecular give the threshold per cycle (Ct) (number of cycles before the flourescen can be detected) and the difference in Ct it used to calculate the amount of the target. this can be plotted onto a standard curve and compared to known standard to estimate the amount of target present
Disadvantages:
- DNA qualtiy obtained from FFPE tumour block can often be poor resulting in poor amplification - fixation of tissues resulting in cross-linking of DNA and proteins, this damages DNA and can result in artefacts
- accuracy of result depends on the quality of the DNA
Describe minisequencing
single base extension of a single florescently labelled ddNTP at 3’ end of a special oligonucleotide complimentary to a sequence 1 base upstream of the examined polymorphic site
- ddNTP are flourescently to enable ID of the incorporated base and hence the complimentary dNTP in the target sequence
Describe OLA?
OligoNT ligation assay
1) PCR primer hybridised to target (designed with WT or mutant 3’ end and a different length stuffer seq for size fractionation)
2) PCR performed
3) ligation reaction- common primer with fluorescent FAM at 3’ end meets the 1st primer over the mutation position in the altered mutant allele. If the 3’ end of the primer matches perfectly with the target, both primers will be ligated. If there is a mismatch ligation wont occur
Describe Pyrosequencing
sequencing by synthesis
for sequencing a sinlge strand of DNA by synthesising the complimentary strand.
- one base is added at a time. If incorporated the formation of the phosphodiester bond results inthe reslease of pyrophosphate.
- this pyrophosphate is used by sulfarase to produce ATP which results in light being emitted by a luciferase catalysed reaction
- light is detected by a camera and the amount is proportional to the number of bases added
- best for short reads
-unreliable for long mononucleotide repeats
What are the different methods for allele sizing?
- flourescent PCR
- TP-PCR
- Southern blot
- long range PCR
- chimeric PCR
- Long range PCR
What are the considerations of sizing PCR?
Native DNA needs to be fragmented (restriction enzyme digest, sonication etc.) otherwise the DNA will be too large to pass through the gel or super-coiled and will pass through the gel at a rate disproportionate to its size.
PCR products do not need to be fragmented but alleles dropout (due to PSPs or secondary structure), preferential amplification of the smaller allele, or lack of allele heterozygosity may hinder estimation of allele sizes
What are the different types of electrophoresis?
Agarose gele electrophoresis
PAGE (polyacrylamide gel electrophoresis)
Pulse field electrophoresis
capillary electrophoresis
Describe agarose gel electrophoresis
- separates fragments of 500bp to 25kb
- during gelation agarose polymers associate and form a network of bundles whose pore size determines a gels molecular sizing properties
1) DNA is applied to Gel
2) electric field is applied and negatively charged DNA migrates towards the positively charged anode
3) speed of migration is inversely proportional to the log of the molecular weight (DNA has a constant mass/charge ratio)
4) DNA can be visualized on the gel using an appropriate dye and size determined by comparison to a size ladder a
the rate of the DNA migration is dependent on the size of the DNA fragment, secondary structure (needs to be denatured) charge applied and the size of the pores in the gel.
Describe capillary gel electrophoresis
Most commonly used method in diagnostic labs
Used in combination with florescent labelled DNA can allow fragments to be sized to within 1bp of each other
1) DNA denatured (heat and DMSO) and ssDNA is passed through a capillary containing polyacrylamide gel
2) rate of migration depends on the size of the fragment and requires an internal size standard to be included with each sample
- amplified fragments can be mixed providing they are of different sizes or have different fluorescent dyes]
Used for MLPA, PCR genotyping, sanger seq etc
Describe the use of fluorescent PCR for sizing assays
e. g. for FRAX, DM1, HD- can detect alleles up to ~5kb depending on template and PCR conditions. it is not suitable to large expansion alleles
- there is preferential amplification of the smaller allele which may mask the presence of a second large expansion allele.
- uses fluorescent PCR primers designed to flank the region of interest (1 of which is flourescently labelled)
products are separated by gel electrophoresis
Describe the use of long range PCR for sizing assays
can be used to amplify fragments over 5kb by the use of PCR additives
- betaine equalises AT and GC contributions to strand pairing
- DSMSO weakens base pariing and destasbilises secondary structure (used for triplet repeats)
use a mix of polymerases
- taq pol is high processivity by low fidelity asd it lacks 3’-5’ exonuclease activity so it can introduce errors
- pfu is a proofreading enzyme that can correct taq errors
Uses:
provide a template for NGS
nested PCR to overcome pseudogene interference e.g. PMS2
-test for large common IKBkG deletion in incontentia pigmentia
Describe the use of TP-PCR for sizing assays
Specialised type of PCR to enable to amplification of large triplet repeat expansion alleles
- 3 primers
A repeat primed primer primes from all the repeats to create products representing the full length of the repeat. It works with the forward primer to do this and is present in limiting amounts so that it is used up quickly, this prevent gradual shortening of the pool of PCR products.
the repeat primer contains a unique sequence not present in the genome. a 3rd primer is complimentary to this and it works with the forward primer to amplify all the repeat primed products
Advantages: periodicity of the peaks will indicate the number of repeats present
-can identify AGG interruptions in fragile X, the presence on interruptions is associated with the risk of expansion on transmission. although this information is not currently reported on UK Fragile X reports.
disadvantages: very large expansions will not be accurately sized but the presence of a peak for the full length expansion will identify samples in the pathogenic range.
- PSPs can result in false results
- interruptions can result in false -ves in DM1 so the reaction is run in both directions to overcome this
Describe the use of chimeric PCR for sizing assays
A type pf TP-PCR that has been used for HD but only uses 2 primers
- forward primer
- reverse primer is chimeric with 2 annealing sites. The 5’ end is specific to the region of the HTT flanking the CAG repeat and the 3’ end is complimentary to the CAG repeat
the 5’ end will anneal at high temp- provind a full length product peak
the 3’ end will anneal at low temperatures to generate the CAG stuttering pattern for allele sizing.
this can detect all CAG alleles and expansions up to 40 repeats
Primers avoid all SNPs and the assay can be used as a standalone test for HD. it overcomes the need to co-amplify both alleles using alternative primers in patients apparently homozygous for an allele in the normal size range (and exclude the risk of an undetected expansion allele)
Describe the use of inverse PCR
Inverse PCR is used to detect distinct genomic rearrangements e.g. Hemophilia A intron 22 inversion:
1) BcII digestion of DNA
2) DNA ligated into circularized DNA
3) primer added for PCR (primers would normally point away from each other in genomic DNA but will form a PCR product when the inversion is present)
4) primers can be multiplexed so both fragments can be amplified in a single reaction
What is southern blot used for in sizing?
Can be used to resolve large fragments to large to be amplified by standard PCR
- currently used for fragile X referrals where the methylation status is also required e.g. prenatals - new Asuragen TP-PCR with methylation status included.
- testing methylation status requires a methylation specific restriction enzyme
- also used for FSHD testing and c9orf72
- labour intensive, not optimised for high throughput testing and requires a large amount of good quality DNA
Describe the method for southern blot
1) genomic DNA digested by restriction enzyme (no need for PCR) to isolate region of interest. a double digest can be used to also test for methylation status
2) DNA + controls and size ladder are applied to gel
3) separate fragments using gel electrophoresis
4) DNA is transferred to a negatively charge membrane by capilliary action and fix to the membrane
5) a labeling probe is applied to label to label the DNA fragment. unused binding sites are blocked by BSA
6) washed to remove unbound probe
7) detect probe and visualize bands e.g. autoradiograph for radiolabeled blots
What are the considerations for methylation detection
Most techniques require PCR amplification but methylation is lost during PCR so DNA need to be modified first so that methylated and unmethylated DNA can still be distinguished
What is bisulphite modification?
bisulphite modification results in the conversion of unmethylated cytosine to uracil which is replicated as a T.
methylated cytosines remain unchanged
can then either use PCR primers that are methylation independent for proportional amplification of methylated and unmtheylated DNA or methylation specific primers which are designed to amplify the methylated target only
How can pyrosequencing be used to detect methylation status?
1) bisulphite conversion
2) methylation insensitive amplification if region of interest
3) sequencing to report presence of T (unmethylated) or C (methylated) at each restriction site
sensitive to ~10% methyaltion but high cycle number required means it is prone to contamination
How can methylation sensitive melt curve analysis be used to detect methylation status?
The higher GC content of uncoverted (methylated) DNA makes it more resistent to melting.
- methylation independent PCR performed in the presence of a dsDNA intercalating dye
- results in peaks indicating melting temp. For a mix of methylated and unmethylated DNA 2 digest peaks are formed. results of a heterogenous mix can be complex and hard to interpret
How can real time quantitative detection be used to detect methylation status?
methylight
- methylation independent PCR and differentiates between methylated and unmethylated DNA using specific taqman probes (reported and quencher) with different flourophores
How do methylation specific primers work?
highly sensitive but they have a high false positive rate due to false priming events (mismatches between primer and template) and incomplete bisulphite conversion
Give examples of MSP techniques
NGS- of bisulphite converted DNA is possible for targeted regions or for the whole genome but is largely used int he research setting
array based methylation analysis- Human methylation 27 (illumina) offers coverage of over 450,000 methylation sites for GWAS studies
What are non bisulphite methods of methylation detection?
methylation sensitiev restriction digest and assay of the digestion
1) southern blotting
2) FMR1 mPCR
3) MS-MLPA
Describe southern blotting for methylation detection
e.g. used for fragiel X and can detect methylation status as well as the size of the expansion
double digest using methylation sensitive restriction enzyme e.g. Eag1 to digest a larger fragment containing the expansion produced by a methylation insensitive EcR1 digest
- normal male alleles will have 1 band from the Ecor1 digest
- male expansion alleles will have a differnt sized band due to the methylation of the FM- may appear as a smear on the gel
- all females will have tow alleles due the methylation of the inactve X
- mosaicism is difficult to assess and incomplete digestion can be an issue
- time consuming and labour intensive compared to PCR
- BPG - do not use DNA from lith hep tubes as this can migrate anonymously in the gel
- methylation pattern may not be reliably set in CVS samplers so AF are preferred for prenatals if southern blot is being performed. Usually only to confirm that a full expansion mutation is methylated following detection of an FM or large PM by TP-PCR in case of methylation mosaicism
Describe FMR1 mPCR for methylation detection
amplidex TP-PCR contains a long range PCR for allele sizing and TP-PCR. In mPCR a methylation sensitive restriction enzyme is also used.
Purified genomic DNA
mixed with control DNA and reaction split into 2 tubes. - 1 tube has a control digestion that it not methylation specific (FAM labelled to detect repeat size)
- 2nd tube has a methylation specific digestion (HEX labelled to detect methylation status)
PCR is performed for both tubes using different coloured flourophores
amplicons are then pooled and seperated by Gel electrophoresis for data analysis
- can determine the extent of methylation of each allele in male and female alleles eliminating the need for S. blot
- methylation data is expressed as a percentage of the undigested DNA
- faster and more accurate than S blot especially for low level methylation mosaics
- if used with the asuragen FMR1 CCG kit can also assay the AGG interruption repeats to give full ascertainment of anindividuals FMR1 alleles.
Describe MS-MLPA for methylation detection
BWS/SRS, PWS/AS and UPD 6, 7 and 14
Same as MLPA except the hybridisationof MLPA probes reaction si split into two tubes.
- methylation sensitive probes contain a HhaI site.
- 1 tube is treated as standard MLPA for copy number determination
- 2nd tube is incubated with a HhaI whilst probes are ligated- unmethylated probes and sample are digested and can’t be amplified in subsequent PCR
- digestion control is included to prevent false negative results
MS-MLPA in tumours
used to examine transcriptional inactivation if a TSG
e.g. MLH1 promoter methylation associated with sporadic CRC
What are the cytogenetic and molecular techniques for CN detection
G-banding FISH MLPA QF-PCR NGS ARRAY
What is G-banding?
Manipulation of the cell to obtain metashase chromosomes. Cells are harvested and then banded using giemsa staining so the banding pattern can be visualised.
- requires actively dividing cells
- to get the highest yield of metaphases cells are synchronize by being stopped in S phase by BrDU
-cells are then released and allowed to passage to Metphase before being harvested ans stained
- Treatment with a hypotonic salt solution just prior to
harvest permits swelling of the nuclei. Incubation in a
dilute KCl or sodium citrate solution for 10–30 min
generally achieves good spreading. Insufficient hypotonic treatment results in chromosome spreads that are tightly knotted; individual chromosomes are difficult to virtually
impossible to visualize.
- last step is to preserve the cells with fix (at this point they contain no risk and can be handled without gloves or safety cabinet). Fixation with Carnoy’s solution, a
mixture of methanol and glacial acetic acid, arrests the
process of hypotonic swelling and all metabolic processes
- slide making is by dropping the fixed solution onto slides. the humidity and height the solution is dropped from affects the chromosome spreading and is optimised for max spread between chromosome so they can be analysed without broken cells
- cells are aged in an oven and can then be stained.
what is G-banding used for?
- follow-up of positive anueploidy screen
- follow-up of abnormal array
- family studies for a known rearrangement
- infertility and POI
- sperm and ovum donors
What are the benefits and limitations of G-banding?
- low resolution
- requires actively multiplying cells
- labour intensive, slow TAT
- can’t detect UPD
- risk of cultural artefact (PND)
- some abnormalities not seen in cultured cells e.g. some mosaic aneuploidies
- aneuploidy detection and can determine recurrence risk
- provides structural information
- can detect balanced rearrangements
- whole genome screen
- can detect mosaicism- sensitivity increases with more cells counted
- relatively robust and inexpensive
describe the use of FISH for CNV detection
uses flourescently labelled ssDNA probes targeted to the region of interest. In the presence of a deletion there will be no signal so also need to include a control probe ensure that hybridisation has occurred. For a duplication there will be extra signal either in the same location for tandem dups which may appear as one brighter signal or elsewhere if the duplication is present on another chromosome.
What are the uses of FISH
mainly used in cancer on FFPE or BM to ID recurrent genomic rearangements for prognostic or treatment stratification.
Aneuploidy detection and microdels/dups (arrayCGH has superseded this for most referrals except urgent family testing)
origin or marker from g-banding.
If G-banding fails e.g. AML screen
gene amp in cancer e.g. HER-2 for breast cancer
post transplant chimerism monitoring
PGD
what are the benefits of FISH
- can be done on interphase (don’t need to be dividing) or metaphase cells (positionl information)
- can scan a lot of interphase cells to detect mosaicism
- rapid FISH can provide a fast TAT (same day if lab aware of sample and ready to set-up)
- can analyse single cells
- higher resolution than G-banding
- probes can be deisgned for alomost any genomic region
- can detect subtelomeric and cryptic rearrangements missed by G-banding
what are the disadvantages of FISH
- can get artefacts fromt he co-localiastion of 2 probes so they appear as one signal
-non-specific binding- - targeted test so need to know what you are looking for
cannot detect UPD
-cannot detect MCC in female pregnancies - limited number of probes can be used at a time due to a limited number of flourophores
- interphase FISH doers not provdie positional information
-micro duplications can be missed due to low result ion on metaphase spreads.
Describe QF-PCR for copy number detection
quantification of polymorphic repeat regions using flourescently labelled primers. Quantification is relative to the other markers in the multiplex, not absolute. PCR reactions is stopped whilst still in the exponential phase where the amount of input DNA is proportional to the amount of PCR products
- it is most commonly used to detect anueploidy in prenatal samples and MCC
- can also be used for UPD (requiresd samples from both parents), post transplant chimerism monitoring and sexing
what are the advantages of QF-PCR
- detects mosaicism to ~10%
- cheap, quick, fast technique
- can be high throughput
- requires very little starting material
- does not require confirmation by another method
what are the disadvantages of QF-PCR?
- does not provide positional information
- targeted not whole genome
- MCC can affect result interpretation but can be identified
- may not detect low level mosaicism
- limited abilit to muliplex sue to limited number of spectral dyes. Cannot use lots of different stuffer fragments as the lengths of the PCR products will affect the efficiency of the PCR amp and result in inaccurate quantification
What is real time QF-PCR?
PCR where the amplification is measured during each PCR cycle using fourescent DNA binding dyes
- SYBR green (non-specific)
- taqman (seq specific)
measure during the exponential phase where the amount of product amplified is proportional to the amount of starting material
The ‘threshold’ number of cycles requires for the fluorescent signal to be detected is called the Ct
However, the efficiency of amplification is often variable among primers and templates. Therefore, the efficiency of a primer-template combination is assessed in a titration experiment with serial dilutions of DNA template to create a standard curve of the change in (Ct) with each dilution.
To quantify gene expression, the (Ct) for an RNA or DNA from the gene of interest is subtracted from the (Ct) of RNA/DNA from a housekeeping gene (usually ABL1) in the same sample to normalize for variation in the amount and quality of RNA between different samples. This normalization procedure is commonly called the ΔCt-method[14] and permits comparison of expression of a gene of interest among different samples.
what are the uses for real tim qPCR
- quantify gene expression (mRNA)
- minimal residual disease monitoring as has high sensitivity (e.g. BCR-ABL)
- detection of mutations against high background of WT - cancer - JAK2, V617F, NPM1, FLT3-ITD
what are the advantages of real time-qPCR?
- quantitative
- high resolution
- high sensitivity
- rapid and easy to perform allowing a fast TAT
- requires little starting material
- post PCR processing is eliminated reducing labor, cost and risk of cross contamination
what are the disadvantages of real time qPCR?
- no positional information
- targeted (not whole genome)
- requires specialist equipment
- unlikely to detect low level mosaicism
- multiple reactions required to examine multiple loci (Not multiplexed)
- non specific SYBR green intercalates with any ds DNA resulting in false results
What are the advantages of MLPA?
- can be highly multplexed
- high throughput
- inexpensive
- requires little starting material
- can detect methylation
what are the disadvantages of MLPA
- targeted
- more labour intensive than PCR
- cannot detect low level mosaicism
- can be used for single cell screening e.g.PGD
- no positional information
- cannot detect balanced rearrangements
- may not get the full breakpoints of a del or dup if it extends beyond the genomic region covered by the kit.
- sensitive to contaminants
- PSP can result in a false result - single exon deletions should be confirmed by another method
describe the principle of MLPA
- dual probes are targeted to region of interest. Probes are in two parts and are designed to hybridise next to each other on denatured ssDNA of interest.
- the probes are ligated by a ligase to produce a continuous probe
- all probes have the same primer seq allowing all probes in the mix to be amplified by a universal primer pair
- probes have different length stiffer fragments which allows them to be distinguished when the products are seperated by capilliary electrophoresis
- use of dual rpbes increases the specificity of the reaction
what are the steps of MLPA?
- denature DNA
- hybridise probes
- ligate p
robes - PCR amp pf probes (only probes that have the target region present will be amplified)
- separate probes by capillary electrophoresis
- analysis
each reaction needs a control to normalise against. Also need positive and negative controls to check for contamination and ensure that the reaction has worked. the controls also aid in interpretation, troubleshooting
expected ratio: 1 - normal 0 - hom del 0.5 (0.3-0.7) - het del 1.5 (1.3-1.7) - het dup 2 - hom dup
How does MS-MLPA also detect methylation?
There are methylation probes included in the mix which contain a HhaI restriction site.
- following probe hybridisation the reaction mix is split in 2
- 1 tube has a standard MLPA reaction and is used to determine copy number
- the second tube is treated with HhaI which will digest unmethylated. Methylation status is detemined semi-quantitatively by comparing the undigested tueb with the digested tube. a digestion control is required to confirm that full digestion has taken place
What is RT-MLPA?
RNA is reversed transcribed to cDNA to allow RNA expression profiling. It is more efficient than northern blotting.
what are the uses benefits of NGS for CNV detection?
very high resolution- single base change and break points
- can be genome wide or targeted
- provides positional information
-detects UPD and LOH
balanced and unbalanced rearrangements can potentially be detected (pair-end and read-mate sequencing)
- SNVs and CNVs can be analysed in a single assay
- WES has lower const and higher coverage but less able to detect structural chages
What are the disadvantages of NGS for CNV detection?
- expensive
- can be difficult to detect CNVs- lots of VUS likely to be detected
- vast amounts of data produed means that storage is an issue (costly) and identifying the data of interest requires a high bioinformatics input
- labour intensive
- not suitable for small genes
- need to confirm abnormal results
- WES may introduce bias from the initial targeting step which would not occur with WGS (PCR bias in amplicon based techniques due to GC content and probe based targeting may result in off target results and difficulty with repetitive regions)
What are the four main methods for detecting CNVs using NGS
- read depth
- duplication results in increased read depth and deletion results in a decrease. This technique requires high coverage to detect imbalances and to distinguish artefacts from real calls
- can detect dels and dups but breakpints are unprecise and cant detect SVs - read pair
uses reads from paired end sequencing with an known distance between the two reads. If there is an increase in the distance between the reads this indicates and insertion and a decrease in the distance indicates a deletion
- can detect most SVs but unable to detect the exact breakpoints - split read
uses reads from pair end sequencing where one of the pair perfectly matches and the other completely or partially fails to map to the genome. The unmapped reads are a potential breakpoint
-can detect exact breakpints but is limited in the length of reads and NGS <1kb affects the accuracy and and precision - de novo assembly and comparison to reference genome. less common as there is a huge bioinformatic demand. Performs badly on repetitive or complex regions
RP and SR are both poor in repetitive regions or regions with segmental dups as it is hard to accurately map reads to the reference
What are the principles of an array?
There are 3 mandatory components to an array:
1) an array containing immobilized allele specific oligonucleotide probes
2) fragmented nucleic acid seq that has been flourescently labelled
3) detection system to record and interpret hybridisation signal
the signal intensity depends on the amount of target DNA sequence is present in the sample and the affinity between the probe and the target
What are the differences/ benefits of SNP array?
- CGH involves running the patient DNA against a sex-matched control
- SNP: - a control DNA is not required as the data is normalised against a virtual cluster file which is composed of 100s of “normal” samples.
- Knowing the sex before had is therefore not necessary for SNP array as it can be predicted using the SNP data.
- For arrayCGH, the sex must be known beforehand to ensure it is run against the correct sex.
- Both are limited in detecting mosaicism but SNP array is more sensitive due to additional data from B allele frequencies.
- SNP array, but not aCGH, can detect copy neutral absence of heterozygosity (AOH). This includes uniparental isodisomy, where both copies of the region are inherited from one parental chromosome - uniparental isodisomy of imprinted regions is associated with imprinting syndromes. AOH, particularly mosaic AOH, may reflect acquired isodisomy, referred to as loss of heterozygosity in the context of neoplasia. SNP arrays therefore more useful in analysis of cancer samples.
- SNP array requires lower input DNA
- For prenatal samples, SNP array can reveal maternal cell contamination through the presence of additional genotypes in the B allele frequency plot. Maternal cell contamination is not detectable by aCGH.
- SNP can detect triploidy (arrayCGH would appear normal as there would be an additional copy of every chromosome and when normalised against each other the log ratio would remain at 0)
- SNP can predict origin of aneuploidies
what are the reporting challenges of SNP arrays?
- SNPs are not evenly distributed in the genome, being more common in non-coding regions, which are typically under less evolutionary constraint. Limited number of SNPs in the genome, which limits array design. Combined SNP/oligo arrays can improve coverage.
- Areas of segmental duplication don’t contain SNPs so less accurate detection of CNVs
- SNP density can be predicted by the presence of microsatellites: AT microsatellites are potent predictors of SNP density, with long (AT)(n) repeat tracts tending to be found in regions of significantly reduced SNP density and low GC content
- When MCC detected cannot tell if the majority profile is fetal or maternal (unless abnormality present) i.e if 10% contamination detected cannot tell if it’s 10% fetus or 10% maternal
- Artefacts due to misbehaving SNPs
- Data is more complex to interpret due to additional data from B allele frequencies. Complex patterns observed when mosaicism is present. Therefore the training and interpretation requirements are more complex
What are the interpretation challenges of SNP arrays?
• Ethical issues
o AOH – AOH is relatively common throughout genome, when/what/how to report? Clinical significance of UPD for most genomic regions is uncertain, may choose not to investigate regions of AOH which do not involve well-establish imprinting syndrome regions
o Safe guarding issues relating to high levels of AOH
o Consanguinity?
o Non paternity
• Difficulties with new tech (regulation, BPG, incomplete reference data for analysis, losing applicability of your local database of CNV)
SNP array may detect CNVs in cancer-susceptibility genes that are not detectable using clinical aCGH platforms which have minimal coverage of these genes. The laboratory needs a policy for validation and reporting of such CNVs, including liaison with Clinical Genetics. Referring clinicians may not be aware of the risk of incidental findings and may not be counselling families appropriately.
What is western blot?
method to separate proteins and can be used to determine the presence or absence of proteins, compare protein levels, assess purity of relative molecular
what are the steps in western blot?
- extract protein- can be from whole tissue or tissue cultures. cells are lysed to release the proteins of interest and protease inhibitors may be included
- separate proteins (SDS PAGE)
- blotting- protein are driven from the gel to a stable support membrane by exposure to an electrical field or a vacuum. - generally nitrocellulose
- block non specific ab binding with BSA
- labeling
- visualization and analysis
Describe SDS PAGE
SDS PAGE is a mtheod to spearate proteins based on their moecular weight. Under denaturing conditions the -ve charged detergent SDS binds to hydrophobic regions of proteins causing them to unfold and dissociate from other proteins. SDS also confers a negative charge to the protein relative to its length, so when a charge is applied to the proteins in a gel they will migrate to the anode with the rate based on their molecular weight.
- non denaturing conditions if protein-protein interactions or folding are being investigated, IN this proteins are separated based on net charge, size, shape as protein carry a net -ve charge in alkaline buffers
what is 2D SDS PAGE?
1- proteins are seperated by isolectric focusing and are run on a gel until they reach their isolecetric point. (pH at which the net charge on the protein in zero)
2- proteins are separated by their molecular wieght.
this allows separation of many more proteins (2000) that standard SDS PAGE (~50)
How are SDS proteins detected?
membrane is blocked to prevent non-specific ab binding using non-fat milk of BSA
- direct detection uses a labeled Ab targetd to the protein of interest
- indirect uses one Ab to bind to the Ag and a second labelled Ab which binds to the Ab
How are SDS proteins visualised?
1) chromogenic methods (low sensitivity) use an enzyme e.g. horse radish peroxidase/alkaline phophatase plus the enzyme substrate. This results in the production of a colored precipitate which can be visualized on the membrane as a colored band
2) chemiluminescence (high sensitvity)- uses a substrate that produces chemi-luminescence and can be visualised on x-ray film.
3) fluorescence using Ab coupled to a flourochrome. less sensitivity but allows multiple targets to be visualized at the same time.
What is immunoprecipitation (IP) and co-immunoprecipitation (CIP) ?
used to enrich for a purify a protein from a complex sample. Purified Ags can then be quantified and anlaysed using ELISA or western blot and novel proteins can be identified by mass spectrometry
what is the method for IP?
The Ab for selecting the Ag of interest can be pre-immobolised (most common) on a solid support e.g. agarose of magnetic beads. The Ag is incubated with cell lysate containing the Ag of interest. Unbound cell lysate is weashed away and the g can then be eluted from the bound Ab
Free Ab approach can also be used. In this case the Ab is free in the cell lysate solution and allowed to conjugate with the protein onf interest. The complexes are then retained by attachement to beads and the remaining cell lysate is washed away. This method is good for proteins present in low concentrations of when there is low Ab-Ag affinity
what is CIP?
CIP is used to detect physiologically important protein-protein interactions by using protein specific Abs to target specific proteins and the bound partners. Protein complexes can then be analysed to ID binding partners and study the kinetics co binding interactions.
pull down assays are similar to IP only the protein is attached to a non-Ab affinity system on solid support.
what is CHIP?
CHIP is an IP reactions where the targeted protein is isolated from cell lysate with the attached chromatin. This allows studies into gene regulation and sites of different protein intersctions e.g. transcription factors, silencers, promoters etc
what are the applications of protein detection methods in genetics?
Investigating protein stability, expression, activity, location and affinity can be useful for functional studies into the pathogenicity of a VUS, In practice this is by research as to specialist to perform as part of routine diagnostics.
e.g. investiagtion of BRCA1/2 half life in proteins containing mismatch variants
What is immunohistochemistry?
IHC is a method to visualise the presence and location of proteins in a tissue but Ab-Ag binding and use of a labelled Ab e.g. conjugated to an enzyme such as horse radish peroxidase
- non specific Ab binding should be blocked by BSA
- detection methods can include enzymes in chromogenic reactions or directly labeled Abs
difference in monoclonal and poly clonal Ab for IHC
monoclonal Abs are targeted to a sinlge epitope of an Ag. they are less likely to cross-react
polyclonal Abs wll recognise multiple epitopes of the same Ag. They are quicker and cheaper to make but more likely to cross react resulting in false binding.
What are the advantages and disadvantages of IHC?
Advantages:
quick, cost effective and provide positional information
disadvantages:
Ab cross reactivity = false +ve
variation in fixation and processing requires use of controls
requires pathologist expertise for analysis and interpretation
mostly qualitative (semi-quantitative)
low throughput
does not detect abn proteins that are still expressed e.g. missense mutations
requires pathologist to accumulate mark slide and ID tumour material for study
What are the uses of IHC?
- HER2 screening for breast cancer as a predictor of response to herceptin
- dystrophin staining in muscle biopsy for DMD
- IHC for MMR genes in Lynch
- IHC for EGFR to predict response to gefitinib in lung cancer
What is mass spectrometry?
Mass spectrometry measures the mass/charge ratio (m/z). It can be used to ID unknown compounds via determination of the molecular weight, to quantify unknown compounds and detemine the moelcular structure.
It can be used to identify proteins in a mixture- determine a.a. frequencies of peptides, identify post–translational modifications, absolute and relative protein quntities in a mix, examine isoform expression , turnover rate and subcellular localisation
How does mass spectrometry woek?
The samples is injected into the mass spectrometer and ionised and accelerated, the ions are separated by the m/z via electromagnentic deflection
the mass spectrun is the m/z rations of the ions in a samples plotted against their intensities
What is tandem MS?
uses 2 mass spectrometers in tandem. 1st is used to separate the mixture and the second to separate fragmented ions. this allows a greater of seperation and can be used to identify metabolites in numerous metabolic disorders e.g. newborn bloodspot screening e.g. MCADD and PRV- very specific method with a false positive rate of 0.05%
what is MALDI-TOF MS
matrix associated laser desorption/tine of flight MS
used for mutation detection
- has been used to profile tissues in situ (tissue sections) for the classification of tumours e.g lung cancer with the distinction of primary metastasis and nodal involvement
what are the advantages and limitations of MS?
- can comprehensively profile proteins without the need to separate them first
- requires a small amount of protein
- high sensitvity and specificity
- high throughput = cost effective and suitable for screening
but ..
- preferential detection of proteins will lower molecular weight
- proteins present in low amounts may be masked by the more common proteins
what are protein arrays the there uses?
Type 1. use protein capure agents e.g. Abs on an array to capture proteins (Ags) to determine the abundance and type of proteins present in a mix
Type 2: arrays contain proteins or peptides and are sued to study protein-protein interactions,
Used to detect disease, disease subtypes or response to treatment based on protein profiling of biological fluids
What is proteomics?
Proteom
What is proteomics?
Proteomics is the study of proteins and their biological functions- the aim is produce a complete map of the proteome including cellular protein localisation, interaction network/complexes, signalling pathways and post-translational modifications
Data is obtained by using a combination of liquid chromatography, and tandem MS. Peptides are identified by matching tandem ms spectra to the theoretical spectra of all candidate peptides represented in a reference sequence protein database
What is RNA based mutation detection used for?
RNA based mutation detection has been used for:
- the detection and charcterisation of gene fusions e.g. BCR-ABL1
- detection of aberrant splicing, allowing the detection of deep intronic sequence changes that would be missed by exon sequencing
what is reverse transcriptase PCR?
RNA needs to be converted to cDNA by reverse transcriptase for use in PCR.
All RNA species in a cell can be reverse transcribed to cDNA
- RNA is RT into a hybrid of single stranded cDNA and RNA. the RNA strand can then be digested and DNA polymerase used to generate a second strand of complimentary DNA
Describe the use of RT-PCR to detect gene fusions
RNA is converted to cDNA by RT
has been used to detect fusion genes found in leukemia for prognosis, diagnosis and MRD. e.g. Used in CML to detect the t(9;22)(q34;q11) BCR-ABL1 fusion following complete cytogenetic response has been achieved with imatinib as the technique is more sensitive, It can also detect impending relapse before it becomes overt
what are the advantages and disadvantages of RPCR for gene fusion detection?
- very sensitive
- requires a small amount of starting material
- difficult to maintain linearity
- sensitive to DNA contamination
- targeted so cannot detect novel gene fusions
- need to classify fusion breakpoints at diagnosis for the technique to be used for MRD
what are the benefits and limitations of mutation testing using RT-PCR of RNA?
- can analyse larger genes in fewer fragments
- can detect aberrant splicing caused by deep intronic variants which would be missed be missed by exonic sequencing
- can be difficult to obtain RNA from the tissue of interest and where the gene is expressed in the isofrom of interest e.g. to test the affect of variants on the muscle specific isoform of dystrophin requires and invasive muscle biopsy
- CMGS (BPG) recommend testing for SNPs of polymorphic loci to ensure that both alleles are present in the cDNA
- PTC can result in no mRNA being present
How can RT-PCR investigate a VUS predicted to affect splicing?
- need to have a thorough undertsanding of the natural tissue specific splicing patterns and isoforms
- need to rule out the possibility of NMD
- need to obtain mRNA from the tissue of interest
can sequene cDNA of mutant mRNA to ID the affect on the sequence and splicing pattern
What is a minigene assay?
Method to enable VUS and splicing pattersn to be investigated without needing a sample from the affected individual so can be useful for investigating mRNAs in tissues that cannot be easily sampled
- variant of interest can be generated by site directed mutagenesis but the test may not replicate in vivo splicing patterns
- can also be used to elucidate cis and trans regulatory elements and other regulators of pre-mRNA splicing in vivo
-
what is the protocol for a minigene assay?
- exons of interest are transfected into an expression vector e.g. HEK293T cells (also need to study normal and a vector control)
- harvest cells and extract RNA
- perform RT-PCR to compare WT and mutant transcripts for differences
- sequence to confirm
what are the clinical uses of minigene assays?
- used to investigate the fole of premRNA splicing in oncogenesis
- identify variants resulting in aberrant splicing
more laborious that RT-PCR and not available in most diagnostic labs but does not require samples of RNA so good for hard to reach tissue isoforms
What is northern blotting?
RNA is separated by size by gele elecgtrophoresis
- RNA is then bound to an solid support and exposed to labeled probes complimentary to the region of interest
- can be used to investigate RNA exporession pattersn, splicing isoforms of genes of interest (based on size)- can identify tissue specific splicing and compare splicing between different tissues, developmental times, in disease and non-disease states
what are the advantages and disadvantages of northern blotting?
- able to detect small changes in gene expression
- detects RNA szie
- can observe all splice products
- samples are degraded by RNAses
- requires harmful chemicals
- usually only suitable for investigating 1 or a couple of genes
What is an RNA microarray?
Array with probes targeted to RNA and is used to measure RNA in order to ID changes in gene expression and commonly used for RNA profiling in humans
- only known gene can be investigated so not suitable for identifying novel genes, fusion gene
Microarrays can also be sued for expression profiling of miRNAs
-miRNAs are involved in regulation og mRNA expression, their expression is tissue specific and critical to the development of organisms and they have been implicated in oncogenesis. Therefore miRNA profiling has been used to ID biomarkers for diagnosis and prognosis in cancer
How does RNA microarray compare to RNA seq
The main difference between RNA-Seq and microarrays is that the former allows for full sequencing of the whole transcriptome while the latter only profiles predefined transcripts/genes through hybridization.
RNA microarray is cheaper, quicker and easier than RNA seq with reduced bioinformatics input
- this results in lower costs, shorter TAT and it can also provide quantitative accuracy
RNA seq
- allows unbiased detection therefore it can be used to ID novel transcripts, gene fusions, SNVs and indels that the array can’t
- broader dynamic range
- higher specificty and sensitivity
- easier detection of low abundance transcripts by increasing the coverage)
- However the bioinformatics requirements are much greater especially for de novo assembly of novel transcripts
What is RNA analysis by NGS and what are the considerations?
RNAseq uses NGS sequencing technologies to analyse the transcriptome
- RNA-Seq facilitates the ability to look at alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/SNPs and changes in gene expression over time, or differences in gene expression in different groups or treatments.[4] In addition to mRNA transcripts, RNA-Seq can look at different populations of RNA to include total RNA, small RNA, such as miRNA, tRNA, and ribosomal profiling.[5] RNA-Seq can also be used to determine exon/intron boundaries and verify or amend previously annotated 5’ and 3’ gene boundaries. Recent advances in RNA-Seq include single cell sequencing and in situ sequencing of fixed tissue
in library prep RNA is converted to cDNA as it is more stable that RNA and facilitates amplification using DNA polymerases
- can also select for RNA of interest by looking for specific features e.g. mature processed mRNA can be isolated by virtue of the poly(a)tail using poly(T) oligomers covalently attached to a substrate, typically magnetic beads
what are the uses of RNA seq?
- evaluate the expression of mutant alleles, detect known and novel gene fusion and splicing variants
- has been used to detect the crytpic ETV6-ABL1 fusion in B-ALL which is missed by karyotype
- has been used to investigate tumour heterogeneity (a key factor in disease recurrence, therapy resistance and growth)
- new method basescope can be used to detect mutation specific RNA by in situ hybridisation e.g can be used to target BRAF, KRAS, PI3KA specific mutations in CRC
what is total RNA seq/ single cell RNA seq?
method to sequence the full RNA in a single cell. Can look at RNA processing events e.g. recursive splicing by looking at read coverage patterns
can compare RNA expression patterns between different cells (different tissue, developmental time point so disease vs non-disease)
