Laboratory techniques Flashcards

Question

Describe the use of TP-PCR for sizing assays

Answer 1

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

Answer 2

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)

Answer 3

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

Answer 4

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

Answer 5

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

Answer 6

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

Answer 7

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

Answer 8

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

Answer 9

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

Answer 10

methylight - methylation independent PCR and differentiates between methylated and unmethylated DNA using specific taqman probes (reported and quencher) with different flourophores

Answer 11

highly sensitive but they have a high false positive rate due to false priming events (mismatches between primer and template) and incomplete bisulphite conversion

Answer 12

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

Answer 13

methylation sensitiev restriction digest and assay of the digestion 1) southern blotting 2) FMR1 mPCR 3) MS-MLPA

Answer 14

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

Answer 15

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.

Answer 16

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

Answer 17

used to examine transcriptional inactivation if a TSG | e.g. MLH1 promoter methylation associated with sporadic CRC

Answer 18

``` G-banding FISH MLPA QF-PCR NGS ARRAY ```

Answer 19

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.

Answer 20

- follow-up of positive anueploidy screen - follow-up of abnormal array - family studies for a known rearrangement - infertility and POI - sperm and ovum donors

Answer 21

- 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

Answer 22

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.

Answer 23

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

Answer 24

- 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

Answer 25

- 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.

Answer 26

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

Answer 27

- detects mosaicism to ~10% - cheap, quick, fast technique - can be high throughput - requires very little starting material - does not require confirmation by another method

Answer 28

- 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

Answer 29

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.

Answer 30

- 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

Answer 31

- 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

Answer 32

- 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

Answer 33

- can be highly multplexed - high throughput - inexpensive - requires little starting material - can detect methylation

Answer 34

- 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

Answer 35

- 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

Answer 36

1. denature DNA 2. hybridise probes 3. ligate p robes 4. PCR amp pf probes (only probes that have the target region present will be amplified) 5. separate probes by capillary electrophoresis 6. 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 ```

Answer 37

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

Answer 38

RNA is reversed transcribed to cDNA to allow RNA expression profiling. It is more efficient than northern blotting.

Answer 39

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

Answer 40

- 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)

Answer 41

1. 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 2. 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 3. 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 4. 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

Answer 42

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

Answer 43

* 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

Answer 44

* 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

Answer 45

• 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.

Answer 46

method to separate proteins and can be used to determine the presence or absence of proteins, compare protein levels, assess purity of relative molecular

Answer 47

1. 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 2. separate proteins (SDS PAGE) 3. blotting- protein are driven from the gel to a stable support membrane by exposure to an electrical field or a vacuum. - generally nitrocellulose 4. block non specific ab binding with BSA 5. labeling 6. visualization and analysis

Answer 48

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

Answer 49

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)

Answer 50

membrane is blocked to prevent non-specific ab binding using non-fat milk of BSA 1. direct detection uses a labeled Ab targetd to the protein of interest 2. indirect uses one Ab to bind to the Ag and a second labelled Ab which binds to the Ab

Answer 51

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.

Answer 52

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

Answer 53

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

Answer 54

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.

Answer 55

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

Answer 56

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

Answer 57

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

Answer 58

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.

Answer 59

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

Answer 60

- 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

Answer 61

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

Answer 62

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

Answer 63

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%

Answer 64

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

Answer 65

- 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

Answer 66

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

Answer 67

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

Answer 68

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

Answer 69

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 1. 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

Answer 70

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

Answer 71

- 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

Answer 72

- 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

Answer 73

- 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

Answer 74

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 -

Answer 75

- 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

Answer 76

- 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

Answer 77

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

Answer 78

- 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

Answer 79

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

Answer 80

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

Answer 81

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

Answer 82

- 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

Answer 83

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)

Answer 84

Sanger sequencing is known as the chain termination or dideoxy method - it involes the use of ddNTPs whihc lack a 3'OH group and cant be extended from. These are flourescently labelled and are used to generate a series of fragments, each terminated at different positions whihc represent the full length of the sequence of interest. the fragments are seperated by gel ecletrophoresis and the sequence is determined by the fkrourescent signal as it pases through the detector

Answer 85

1. PCR amplify target DNA 2. run products on gel to check that the amplification has been successful and to rule out the presence of contamination (blank) 3. clean up the sample to remove any unincorporated dNTPs 4. sequencing the sample- each ddNTP is labelled with a different coluured flourophore. ddNTP present in limiting amounts - sequencing involves strand separation, primer annealing to ss template, polymerase extension (more dNTPs so these will be incorporated the majority of the time), random chain termination when a ddNTP is incorporated. this results in a collection of different lenght fragments

Answer 86

the fragments are separated by gel electrophoresis- as the fragments of different lengths pass the detector the flourophore is excited and the flourescence is detected by a detector to create an electropherogram

Answer 87

Becoming replaced by NGS for diagnostic sequencing as more genes and greater depth can be analysed with NGS - still considered the gold standard and is used to confirm NGS findings, for gap fill of regions poorly covered by NGS or to look for targeted mutations e.g. PND, PST and carrier testing

Answer 88

- can produce reads of 800-1000bp (NGS is limited to 40-400) meaning it can resolve repetitive repeat regions which can be problematic with NGS if trying to aligen a repeat larger than the read length of the NGS software - requires more template DNA than NGS - requires less computational tools to analyse and store data - better at indel detection and excluding psueodgenes from analysis than NGS - variants under primer binding sites can result in false negatives and allele dropout so need to do SNp checks when designing primers and use a positive familial controls when looking for familial mutations - NGS is better at detecting low level mosaics by increasing sequencing depth. In sanger the limit of detection is 15-20%

Answer 89

All short read sequencing requires preparation of DNA or RNA libraries- the DNA is fragmented and adaptors are ligated to enable sequencing. For single gene seq, panels of WES there is also an enrichment step to select for the regions of interest. Enrichment is not required for WGS

Answer 90

Hybridisation based enrichment or amplicon based enrichment | - choice depends on cost, number of targeted regions, sample type e.g. blood or FFPE, TAT

Answer 91

Amplicon based enrichment uses long range PCR to amplify and enrich for target regions. The PCR primers are designed to be specific for the ROI. Sequencing ready libraries can be produced from these fragments using techniques such as Illumina’s Nextera, or random shearing methods, prior to ligation of adaptor oligonucleotides for sequencing.

Answer 92

Illumina Nexetera - provides library prep from PCR amplicons >300bp. Transposomes are used to randomly fragment the amplified DNA whilst simultaneosly ligating oligonucleotide adaptors to the end of the frgments. A limited PCR cycle is then used to add indexes and full adaptor sequences to the tagmented DNA ready for sequencing

Answer 93

This method is designed to reduce bias and artefacts introduced by amplicon based library prep. this is acheived by the use of UMIs (unique molecular indices). Each DNA molecule has a different UMI Total genomic DNA is fragmented, end repaired and A-tailed in a single reaction. The fragments also have a UMI, sequencing adaptor and index ligated. Target enrichment is perfromed using PCR primers specific for the regions of interest A universal primer can then amplify the library and add further sample indices. Post sequencing, sequencing reads with different UMIs represent different sequencing molecules, those with the same are PCR duplicates and can be removed. the No. of reads for a region with different UMIs can also be used to determine the copy number

Answer 94

Advantages: - low cost - requires less staring material - useful for small regions of interest - can be used for FFPE with appropriate primer design Disadvantages - allele drop-out can result in false -ves. Regions with zero coverage are generally difficult to amplify because of high GC content or suboptimal PCR amplification conditions. - amplification may introduce bias and artefacts due to sequence content (GC) - difficult to multiplex to study larger target regions - readepth cant be used for CNV detection (except QiaSeq)

Answer 95

DNA is fragmented--> tag fragments with sequencing ready adaptors and barcodes--> regions of interest are targeted by hybridisation to sequence specific probes. - oligo probes are specific to regions of the ROI to result in tiling of the complete ROI. Clean-up of the targeted DNA is aided by the use of oligonucleotide baits conjugated to biotin. These can then be sdelcted for by incubating with streptavidin coupled magnetic beads (biotin and streptavidin bind)

Answer 96

Agilent sure selecgt gDNA is sheared randomly to produce small fragments-- barcodes and adaptors are ligated--library is incubated with biotinylated RNA baits (RNA probes are 120nt log to provide high specificity)-- targeted regions are enriched for using streptavidin coupled paramagnetic beads-- target regions are amplified and loaded onto the sequencer

Answer 97

Unique method using restriction digestion for the initial fragmentation stage. This means no specialist equipment is required and there is less DNA required however the use of restriction enzymes means that all fragmentation sites are identical so duplicates can't be removed from the data in the post sequencing processing. The method is also prone to allele dropout is there are variants in restriction sites and small (1-10bp) gaps are often found in the resulting data due to the positioning of the cut sites which cannot be altered. 1) DNA is digested by a double restriction enzyme reaction to produce non-random fragments 2) fragments are captured by hybridisation with biotinylated probes and captured with strptavidin magnetic beads 3) frgaments are ligated to from a closed circle including PCR primer binding sites, barcodes and seq adaotors 4) PCR is performed using a common primer to generate a linear library of enriched fragments. The primer binding sites are designed to be in opposing directions so that only closed molecules will be amplified, therefore increasing the specificity

Answer 98

- more uniform coverage of target regions - can be used to ID CNVs - PCR duplicates are easily removed during analysis to reduce artefacts - useful for large genes panels/ exomes - suitable for FFPE tissue - more expensive, and greater prep time than amplicon methods - high quantity of starting material required.

Answer 99

Illumina (nextSeq, miseq, hiseq) Uses sequencing my synthesis on a flow cells using 4 colour flourophore chemistry flow cells is composed of a flat glass slide with 8 microfluidic channels each covered in covalently attached adaptors complimentary to the library adaptors 1) library fragments are applied to flow cells and bind to adaptors 2) clonal cluster generation is perfromed using in situ bridge aomplification= attached fragment bends over and attaches to a second oligo forming a bridge-- polymerase synthesises a complimentary strand- both strands released and process is repeated 3) after cluster generation there is sequencing by synthesis.- nucleotides have a reversible 3' blocker attached so only one can be added at a time by the pol. All 4 nts are washed over the cell at the same time, each with a different flourescent label, the complimentary base to the template is added and the chain is terminated. After each round of synthesis flourescence is detected by a camera. A de-blocking reaction is then carried out to remove the 3' blocker and the process is repeated until the complete template has be sequenced - clusters mean that there are 1000's of copies of the same fragment being sequenced in the same cluster so the signal is strong enough to be detected sequecing for millions of cluster is carried out at once. During analysis regions of overlap (contigs) between clusters is used to line up the sequences and then can be compared to a reference genome for variant calling.

Answer 100

Based on detecting the release of H+ when a nucleotide is incorporated which can be detected based on the resulting pH change uses a chip with a high density of micro-machined wells, each holding a different DNA template. Beneath the well is an ion sensitive layer to detect H+ pH changes 1) library fragments are clonally amplified onto sphere particles by emulsion PCR (droplet PCR) 2) template containing beads are enriched using a magnetic bead based process 3) sequencing primer and DNA pol are bound to templates and loaded into the sequencer 4) The 4 dNTPs (unlabelled) are sequentially added, if incorporated H+ is released during the formation of the DNA phosphodiester bond- more H+ is released for homopolymer tracts 5) unincorporated dNTPs are washed off and the next is added until the full fragment has been sequenced 7) H+ is detected by a semi-conductor and converted to a voltage

Answer 101

Most commonly used platform high yeild performs well in homopolymer regions expensive sequencer (compared to ion torrent)

Answer 102

fasted TAT on market low cost flexible, scalable with a range if chips relatively poor performance in homopolymer regions high seq errror rate

Answer 103

sequencing by synthesis 4 nucleotides are added sequentially by DNA pol which results in the release of pyrophophate which is converted into a chemiluminescent signal in a luciferase reaction (pysrosequencing) Fragment genomic DNA; add forward and reverse adaptors; emulsion PCR to clonally amplify library on bead Advantages - Longer reads improve mapping of repetitive regions. Good indel detection. Fast run times. Disadvantages- high reagent costs, high error rate in homopolymers and low capacity

Answer 104

SOLID sequencing - sequencing by oligo ligation and detection (not seq by synthesis) - sequencing by ligation of a flourescently labelled hybridisation probes to deduce the signal of 2 bases at a time gDNA is fragmented, coupled to magentic beads and amplified by emulsion PCR (clonal amp). there is 3' modification of the bead and covalent attachment to a glass slide 2 base decoding provides inherent error correction but there is under representation of AT and GC rich regions.

Answer 105

WGS is whole genome including introns and mtDNA WES is only the coding region of the genome- 1-2% of the whole genome but accounts for 85% of known disease causing mutations

Answer 106

- allows analysis of SNVs, CNVs, indels in the whole genome including regulatory regions e.g. promoters and enhancers - enhanced detection of CNVs and structural variation due to more reliable and uniform sequence coverage- differences in PCR efficiency in different regions of the genome (amplicon) or efficiency in probe hyb or non specific binding in repetitive regions (hybridisation) can result in regions of the genome with little or no coverage. Regions of the genome with low sequence complexity have poor coverage as it is harder to design specific capture baits- this can result in poor coverage or off target effects - PCR amp is not required reducing library prep time and the possibility for GC bias - sequencing length is not a limitation. For Wes most target probes are ~120nt meaning it is pointless to sequence longer read lengths - lower average read depth required to achieve the same breadth of coverage as WES - doesn't suffer from reference bias- hybridisation probes/baits tend to preferentially enrich reference alleles at het sites resulting in fale SNV calls - n WES the refseq is targeted so only targeted exons that have been identified so far will be captured. With improved WGS our understanding of the exome will improve

Answer 107

- lower price (although cost of WGS is decreasing faster) as only targets exomes so less sequencing required to cover a target regions at a higher depth and there is less data produced for storage band analysis - reduces data fatigue- fewer variants to interpret than WGS (most of which will not be clinically significant) - reduced cost means it is more feasible to study more samples and can be applied to population based studies

Answer 108

traditional methods involved positional mapping and then sanger sequencing of the region to ID new genes and pathogenic variants (association sudies, linkage analysis, karyotype) - these methods struggle with rare diseases due to the small sample size of affected individuals GWAS has contributed to the discovery of disease loci involved in complex traits where each variant only contributes a small fraction of the observed heritability. Need very large sample sizes for significance to be proven with low penetrance loci NGS- allows a single step approach but presents a challenge in terms of variant interpretation - can sequence and filter variants to ID a shared gene in a cohort of patients with the same phenotype - can sequence and filter for the same variant in a family with the same phenotype - can sequence parent/child trios and filter for de novo or homozygous/compound het variants MDT and a good relationship with clinical genetics is required to determine how to deal with IFs and VUS

Answer 109

- Rare in a population - located within a protein coding gene - directly affecting the function of the protein e.g. frameshift, +1/2 spice variants - follow the observed inheritance pattern in the family (AD, AR, XL) strategies also depend on the pedigree structure, extent of locus heterogeneity

Answer 110

- when the patient has an atypical presentation - high genetic heterogeneity for features - would be slow and time consuming to test all the candidate genes - difficult to confirm diagnosis using clinical criteria or other testing e.g. biochemical

Answer 111

- there is a greater chance of detecting IFs if the whole exome is targeted. This may be beneficial as it may allow the family to access early screening or treatment e.g. for cancer genes but there needs to be specific lab policies in place and the possibility of IFs should be covered in pre-test counselling - there is also a greater chance of detecting VUS- need policy on how these will be reported and whether follow-up testing can be carried out to help confirm if the finding is pathogenic or not

Answer 112

- custom target enrichment (many private companies off specific target kits for cancer exomes of can offer custom designed target capture) These have a lower cost and allow greater coverage including sanger gap fill of regions that are hard to sequence by NGS. this also helps with providing higher quality calls and reducing the need for sanger confirmation which is beneficial in a clinical service - only genes with known disease association are included reducing the interpretation load and potential for IFs alternatively can perfrom full exome seq or clinical exome seq (small gene subset only including those where the gene disease association is known). Following sequencing a virtual panel specific to the phenotype presentation is applied. This is beneficial as the panel can be updated when new disease associations are confirmed and all samples can be treated in the same way in the lab set-up regardless of referral reason. But there is more data generated which needs to be stored and the depth of coverage is sacrificed for a greater breadth so not all genes will be covered to a diagnostic level which may result in false -ve results

Answer 113

- slightly cheaper - better coverage and can gap fill genes with sanger - easier to fully interpret the significance of each variant - fewer IFs - lower requirement for bioinformatic filtering

Answer 114

WES can be used for tumour profiling - has a higher sensitivity than exitisting methods using deep sequencing (500-1000x) - allows parallel testing of multiple samples - can detect any mutation (within coding region etc) in the targeted gene rather than just targeted mutations as is the case for ddPCR and other tumour profiling techniques - short TATs possible with small sequencers e.g MiSeq MRD monitoring - can ID multiple variants to study the emergence of clonal dominance, emergence of new resistance mutations

Answer 115

Now offered as a rapid service in prenatal testing for severe abnormalities detected on USS, following normal PCR- based on results of the PAGE study and shows a better diagnostic rate than just P CR and arry NGS techniques used for NIPT and NIPD- MPS provides the coverage to ID small increases in obverall CN for chr 13, 18 and 21 for NIPT aneuploidy testing

Answer 116

chromatin immunoprecipitation. technique for genome wide profiling of DNA- protein interactions e.g. binding sites of DNA binding protiens, chromatin modifiers, translation start sites, epigenetic mechanisms, CHIP Abs are used to select for specific proteins which are enriched for from the mixture with the associated DNA sequence. The DNA can then be sequenced to ID the specific sequence motifs associated with the protein interaction. Studies e.g. ENCODE and FANTOM5 have been used to reveal the genome wide profiles and binding sites for a range of DNA binding proteins.

Answer 117

generate a cDNA library from RT-PCR of total RNA or can select for a specific RNA seq e.g. mRNA which is polyA +ve - the sequencing data can than be mapped to a ref genome or use de novo assembly. Used to detect novel framshift and splicing variants, map 5' and 3' gene boundaries, novel splicing isoforms, post translational modifications - often used to ID differences in disease states, different tissues or developmental stages

Answer 118

- Not limited to detecting known transcripts - can reveal the location of transcript boundaries to a single base resolution - can accurately quantify expression levels using qPCR and spike in RNA controls - high throughput - offer single base resolution and digital gene expression at the genome scale and is often cheaper than tiling array of large scale sanger seq

Answer 119

- snmall RNAs e.g. miRNA and siRNA can often be sequenced directly but larger RNAs require fragmentation first which can introduce bias - need to decide whether to producde strand specific libraries which have the advantage of yeilding infor about the orientation of the transript which is useful for transcriptome assembly

Answer 120

Cancer genome atlas andf international cancer genome consortium are using DNA and RNA seq to improve the molecular subtyping of cancer and indeitfy novel fusion gene NGS has also be applied to testing circulating tuumour DNA (ctDNA) to identify biomarkers for diagnosis and prognosis. This has an increased sensitivity compared to tumour biopsy and is less invasive e.g. FDA has approved testing for SEPT9 methylation status in ctDNA for CRC

Answer 121

low density clusters can give high quality data but low depth of coverage high cluster density gives a greater depth of coverage but lower qulaity date. If the clusters are too dense the data can be hard to read and information is lost

Answer 122

sensitivity, specificity, reproducibility and need to down sample to test the limit of detection. If not you cannot be sure that you will detect all the variants at the limit stated on the report e.g. 20x or 30x

Answer 123

Base call per cycle file. It is a binary file containing the base call and quality score for each tile in each cycle. This is produced by illumina and is converted to a FASTQ for bioinformatic analysis

Answer 124

text based format for storing nucleotide sequence with base scores. Generally this is the input file for most bioinformatics pipelines

Answer 125

Binary alignment map (stores data in binary format) Formed by aligning FASTQ files to a reference genome using an alignment algorithm can be used in analysis to visualise variants of chekck quality and or coverage of a region

Answer 126

ultra condensed version of BAM file and are likely to become more common in a n effort to reduce the datas storage requirements of NGS tests

Answer 127

variant call file- used to show variants compared to the reference genome. Produced by applying variatn calling to a BAM file - very basic and contains: chrom, pos, ID, ref, alt, qual and filter

Answer 128

FASTQ1) QC check to ensure the base calls are of sufficient qualty. Poor quality calls are removed from the data to prevent false results. In general a phred score of >30 (i in 1000 or 99.9% accuracy) is considered sufficient 2) variant calling 3) annotation- adding additional information e.g. gene symbol FASTQ files can be treated as the raw data input to be processed by the software

Answer 129

To get an overview of the inherent quality of the sequencing data. It is necessary to improve alignment, remove poor quality reads and artefacts looks at per base quality scores, per sequence quality scores, seq duplication scores, over represented sequences.

Answer 130

plots each short read against the reference genome to enable variant calling. Uses sequence alignment algorithms to align the sequencing data and some deviation is allowed to allow to variation. Previously used BLAST which uses high scoring segment pairs within the query seq and target seq - but the algorithm can be inefficient with dealing with the large scale data NGS produces

Answer 131

Currently used algorithms use filtering or indexing to reduce the time and memory required for filtering. - filtering excludes the parts of the genome where no match can be found and can operate a K-mer or pigeon principle - indexing pre-processes the data making scanning more efficient and is used with a Borrows- Wheeler Transform

Answer 132

illumina has a relatively low error rate and is more likely to have mismatches than indels. Indels are rare overall but have a high miscall rate in illumina chemistry when called (0.1%). The main complication arises from the synthesis process being desynchronised between different copies if the DNA template in the same cluster

Answer 133

Different sequencing technology is prone to different types of errors- therefore the error model used for aligning should be chosen with this in mind for maximum acurracy - Hamming distance- calculates the no.of positions where bases are different between the ref genome and sequence - exit distance calculates the number of operations required to convert seq short read data into an exact match of the reference genome

Answer 134

1) pre-processing- refine data, remove duplicates, re calibrate quality scores. (can be part of or separate to the variant calling algorithm) 2) variant calling- often split into SNV, CNVs, SVs and indels- each may require a different specific algorithm for maximum accuracy - alleles observed in 20-80% of reads are called as het - alleles in >80% of reads are called a homozygous this method is affective for bases with a high seq depth and phred score >20

Answer 135

Poor quality sequencing requires more stringent filtering and can miss het calls so probablistic models using bayes are used to calculate genotype liklihood and confidence scores- this improves accuracy indels are harder to call and are very difficult to ID if they are larger than the sequencing length i.e.>~150bp

Answer 136

Annotation adds additional information to the variant call file- gene symbol, transcript no. HGVS nomenclature, variant consequence repeat regions can be identified to facilitate annotation- may be masked

Answer 137

over or underclustering errors depend on the signal-noise and can be affected by corss-talk with nearby clusters, homopolymer count, incomplete extension and the position at the end

Answer 138

main problem is the variance in signal intensity for homopolymers and they can be called inaccurately if they are large. Also have a high error rate in insertion and deletion calls.

Answer 139

A phred score is the log sclae of probability of an incoreect call for any base. It is an importnat tool for rejecting low quality reads, triming low quality calls and determing a consensus sequence between all the sequencing data and variant calls phred10 = 1 in 10 probability of an incorrect call (accuracy of base is 90%) phred 50 - 1 in 100,000 probability of an incorrect call (asccuracy of base call is 99.999%) in general a phred score of 30 is considered good quality for base calling = 1 in 1000 probability of an incorrect call and 99.9% acurracy

Answer 140

Longer read lengths give more information in the relative position of a specific base pair. they are useful for repetivtive or homolougous regions as there is more chace one end will be anchored in a unique region to facilitate base calling more expensive and not high throughput so not available on the NHS. Nanopore technoology is relatively affordable but the error rate is too high for diagnostic use.

Answer 141

Paired end sequencing sequences a fragment from both ends. Because the distance between each paired end is known this information is used by alignment algorithmsto increase the accuracy of alignment. It can also help ID deletion and duplications if the distance deviated from what is expected. It is also better and resolving SVs, indels and inversions. in single end reads the fragment is sequenced in one direction. The process is cheaper and less time consuming and is used when the accuracy of paired end seq is not required.

Answer 142

limited in repetitative regions and regions of shared homology e.g.pseudogens or highly related gene families. Sanger seq is better at alignment as the sequencing length is longer

Answer 143

depth is the amount of time a single base is sequenced. The data is used together to develop a consensus sequence so the more times a base is covered the greater the accuracy. Generally diagnostic labs validate pipelines to detect variants with 20-30x inadequate coverage will result in false -ves

Answer 144

It is essential to asses a pipeline against a truth set e.g. genome in a bottle to asses the sensitivity and specificity of a panel - can also run known to asses the ability to detect more complex variants e.g. indles and CNVs or to detect clinically significant variants in complex regions e.g. GC rich regions. use prevuiously tested samples are +ve controls (sanger seq is the gold standard)

Answer 145

sensitivity should be >95% with a CI of 0.95 | Should be calculated from 3 independent runs to determine the reproducibility (BPG)

Answer 146

sensitivity is down to 5-10% form most SNVs which is OK for inherited disease but may miss some low level mosaicism and may not be sensitive enoughb for oncology samples where there is a high background of WT (hence tumour profiling is usually to a much higher depth 500-1000x) systematic errors increase with increase coverage - use of overlapping paired end reads can increase sensitivity or unique identifer tags

Answer 147

homologous regions - e.g. pseudogenes can result in mismapping and false -ves/+ves repetitive regions- unique flanking sequence is required for accurate mapping. regions longer than the insert region cannot be accurately mapped GC rich regions are not reliably interpreted as there is higher background noise. Most due to the propensity to form secondary structures or accumulate G C flourophores after washing- illumina seq is prone to substitution errors in GC rich regions.

Answer 148

base calls --> demultiplexed to form FASTQ files--> adaptor triming to give analysis ready FASTQ --> alignment to reference genome to giev BAM files --> process BAM to get QC stats ---> variant calling ---> variant anotation

Answer 149

``` Target enrichment Gene selection Transcript selection Design Poly checks ```

Answer 150

barcoding allows samples to be pooled which increases the throughput and reduces the cost per patient. This is possible with exomes and targeted panels as there is far less DNA per patient to be sequenced DNA quality checks are required for NGS as the test is expensive to run. Quality checks look to see if the library prep has enriched for DNA with the correct fragment size and can be by running samples on a gel to check for products of using a tape station

Answer 151

Amplicon based methods - can suffer allele dropout due to rare polys - not scalable - agilent haploplex uses restriction digestion so all fragments have the same breakpoints. fragments are tagged with biotin, sequencing primer motif, PCR primer, barcode and are then cricularised (only circularised fragments are amplified to increase specificity). The circularised fragments are captured by streptavidin beads and PCR amplified. The inclusion of barcodes allows the removal of duplicates after sequncing, increases sensitvity for low freq variants and allows CNV detection which is not possible for other amplicon based techniques Hybrid capture probes/baits e.g. agilent sureselect - difficulty in homologous regions or repetative regions, this can result in off target binding - can be used for CNV detection - easier to have as high throughput with multiple target regions e.g. whole exome - more expensive

Answer 152

clinical scientists/geneticists select genes with published evidence of association to disease - dutch labs have developed a core gene list for consistency - in the UK PanleApp is used. This is crowd sourcing tool to allow virual panels to be developed and shared within the genetic community. Gene reviews are added and genes can be rated from green (high evidence and variants in gene should be reported to patient) to red (low evidence and not suitable for reporting at this time) diagnostic yeild of NGS should be as good as sequential sanger seq. Therefore need to consider if there are pseudogenes present in the panel. These can interfere with sequencing and variant calling in the natural gene and sanger gap fill may be required. limiting the number of genes in a panel increases the coverage of targets so there is higher senstivity for mosaic/ low freq hets Most diagnostic labs opt for sequencing a full exome and application of a virtual panel as there is flexibility in the genes that can be analysed and the data can be re-analysed in the future if new evidence linking a gene to disease is published. This technique is also more suitable for batching as all patients are treated the same regardless of phenotype

Answer 153

Alamut is a useful tool for identifying transcripts- can see which contains all the exons in the gene and what alternative transcripts may be present NGS validation should include justification for the transcript selection- if there are 2 unique transcripts with different exons both should be included.

Answer 154

LRG project aims to provide stable transcripts for universally accepted standards for reporting Each LRG contains- stable transcript with the most up to date biological info (mapping info, anotation of all transcripts, legacy exon and a.a. numbers

Answer 155

Once the genes and transcripts of interest have been determined these can be used to develop a BED file (can use agilent SureDesign) of ROIs with there genomic coodinates. These can be tiled with RNA baits/probes. RNA bait coordinates should be checked in alamut to confirm that they cover the full ROI e.g. exons +/- ROI pseudogenes can sequester some baits resulting in poor tiling so this should be considered in design and some sanger gap fill may be required for full coverage.

Answer 156

Need to consider if they will result in allele dropout during library prep list of common polys (>1%) can be obtained from gnomad and used to filter data post sequencing - removed from variant prioritsation list as unlikely to be causative (need to be careful of some high freq AR variants whihc may be present at a level higher than this e.g. F508 in CF)

Answer 157

``` ACGS BPG outline the requirements for: validation EQA and QC report format pricing patient pathways ```

Answer 158

Validation is required for all new tests to provide evidence that the test will provide the correct result on the sample. It should be carried out for all aspects of the NGS workflow including library prep, sequencing and data analysis including variant calling. it is important to understand the technical limitations of the test so they can be addressed during validation and if required additional tests included in the service e.g. sanger gap fill of regions with pseudogenes. Validation covers the sensitivity (ideally 95% with CI of 0.95), specificity and reproducibility and should identify the optimum conditions for the test to be run at e.g. if DNA conc or volume of consumables used is being changed from manufacturer guidelines

Answer 159

IQC is concerned with internal qualit parameters. These are tested as as indicator that the test is meeting the required standards set out during validation - includes cluster density, no. of reads, coverage - there should be robust reporting and checking of these metrics so that sub-optimal data is not reported, e.g. low coverage may risk het variants being missed. - technical data should be included on the technical report EQA checks the performance of the lab against BPG and other labs. There is a GENQA scheme for germline and somatic testing and involvement is a requirement of accrediation - wet based: gDNA is distributed to the lab so test the technical processing as well as reporting - dry based: FADTQ files supplied to test the bioinformatic processing and results interpretation

Answer 160

- for labs that do not have a robust tube transfer checking system (barcodes or witnessed transfer) to confirm the ID if all samples at all stages BPG require any reported variant to be confirmed by sanger seq on a new DNA dilution - in practice most diagnostic labs have this in place. However variants have been confiremd as the sensitivity and specificity of some tests was not 100%. As NGS has become more commonplace many labs have now tested enough samples to prove that they do not require sanger confirmation for simple SNVs (100% specificity) (may still be required for CNVsd and indels), therefore sanger confirmation is not always required saving time and money. However by not sanger confirming it may just mean that primers need to be designed at a later date for family testing.

Answer 161

The aim of a report is to clearly communicate the genetics results the clinicians. It is split into scinstific and technical sections the scientific section should follow BPG and include: patient identifiers, reason for referral, headline result and result interpretation linked to the patients phenotype. It may also include reauest for parental samples of the offer of PND following counselling where appropriate. Results should be given using HGVS nomenclature (including transcript) and UK labs should be moving to using build 38 by the end of 2021. For negative reports the expected diagnostic yeild of the test should be reported technical section to include: supporting infromationa bout the panel including: genes tested with refseq ID and OMIM no., info on splice sites and primoter regions and info on ehihc regions ahve an have not been covered (especillay if variants in regions not covered have been associated with disease) - methods used (library prep, seq tech, bioinformatic pipeline and software) coverage achieved should be highlighted including regions of poor coverage Vous with clinical relevance may be reported depending ona labs individual guidelines. In general 'hot' class 3's are reported. IFs are reported depending on the labs policy e.g. ACMG59 details of whether dosage analysis has been included

Answer 162

the cost of NGS technology is constantly decreasing however the cost of reagents, and technical and scientist time remains constant. batching patients using barcodes allows more patients to be tested per run but reduces the coverage and is limited by the library prep and platform used. the cost of NGS panel needs to be competetitive.In generall if a pateint has a hetergenous disrder it is cheaper to do the NGS panel then sequential sanger seq and can significantly reduce the diagnostic odessy for the patient. For virtual panels there is the added benefit that further panels can be analysed if the first is negative

Answer 163

- understanding human genetic variation and its association with disease risk - individual response to treatment - translation of genetic data to clinical decision making

Answer 164

Clinical utility & the interpretation of results- the ability to interpret genetic data lags behind the ability to obtain it and not all genetic data is clinically useful so should it be reported? detection of VUS and IFs and variably penetrant alles require expertise in their interpretation and reporting- this can be difficult for novel variants where there is a lack of clinical or functional research to confirm or refute a diseases association. there is a fundamental need to share genetic information between labs to support the interpretation of variants- this is supported with platforms such as decipher. Lab may also have internal variant databases so that they can be referred to is the same variant is detected again.

Answer 165

risk of detecting IFs and VUS-m this is more likely the larger the panel genetic results may also have impact on family members who will not have been counselled or agreed to testing.

Answer 166

Single gene testing is cheaper and there is a lower risk of VUS. But may become more expensive if lots of single gene tests are required to reach a diagnosis panel offer an improved detection rate but there may not be sufficient data in the medical literature to report variants. Improved detection rate needs to be weighed against increased risk of VUS compounding this is genotyping errors and some previously detected variants will have bee misclassified. A study in HGMD showed that 27% of a wide selection of entries where actually benign polys or mis annotated.

Answer 167

usually ascribed to environmental factors or genetic modifiers - correct management may need to bear in mind a patients medical and family history

Answer 168

needs to cover the infor that can be derived from the test and the limitations, the benfits and possibility of IFs and VUS - genes included - whether the report will be added to the patients medical records (issue in the US where there is a fear of insurance discrimination) - whether IFs will be reported - if Trio testing whether the parents want Ifs to be reported if not detected in the proband

Answer 169

the result of the test and the implications for the individual and their family - needs to be a mechanism for re-review of VUS

Answer 170

100K project as well as trying to ID variants in cancer and DD was used to pilot different reporting and testing techniques and act as a proof of principle for the roll out of NGS testing in routine diagnostics. In the 100K project researchers did not have an obligation to search out Ifs (as is the case in the US) as this would distract from the aim of the project, however if one was identified it would be reported if it was considered to be clinically valid (consistent clinical significance) and clinically useful (is knowledge of the variant likely to benefit the parent/family)- these needed confirmation in an accredited lab prior to reporting. They were also carefully considered in conjunction with a clinical geneticist.

Answer 171

single molecule seq aims to sequence a single DNA meolcule without the PCR based amplification or the need to halt between read steps. Therefore there is no reliance on a clonal population of amplified reads or chemical cycling

Answer 172

second generation sequencing is produced by short read technology. IN contrast 3rd generation seq uses long read tech (>10,000bp) therefore there is improved detection of SVs, CNVs, and repetitive regions- these are all important in development, evolution, adaption and disease

Answer 173

- smaller amount of starting material required - higher throughput - simplified template prep lower projected per base cost longer read lenghts (10,000-100,000bp) resulting in enhanced de novo assmebly, haplotype detection, CNV and SV detection, detection of insertions and inversions, detection of balanced translocations and novel chimeric fusion transcripts - enhanced sequencing of repetitive regions resulting in a more contiguous reconstruction of the genome - more uniform covergae - less sensitive to GC content - potential to detect epigenetic modifications e.g. methylation

Answer 174

1) sequencing by synthesis (SMRT - pac bioscience and FRET seq- life technologies 2) nanopore - biological or synthetic (Oxford Nanopore) 3) Syntheic long read technology (Illumina and 10x genioomics)

Answer 175

Conitnuously moniros the incorportation of a differently labelled nt. In second generation seq there is a blocker so the seq is stopped and visualised after each nt hasd been added. Each NT carries a differently coloured florophore attached to the phosphate group. The flourescence is read (laser and camer read the flourescence emitted) as the nt is incorporated and the flourescnet group is naturally cleaved by the polymerase when the next nt is added. sequencing is performed on SMRT cells wihihc have 1000' of ZMWs (zero magnitude waveguide) - this is a metal film with a small hole (30-70nm) in, this means only light can pass through the hole in the bottom of the ZMW so it can detect the activity of a sinlge molecule against the background of 1000's of labelled nt

Answer 176

1. template molecule is circularised by attachment to hairpin adaptors 2. single circularised DNA molecule is attached to the base of each ZMW 3. DNA is processed by the po 4. fluorescent nt are flooded on top of the ZMW 5. when a complimentary nt is included the enzyme holds it is place fractionally longer than an unincorporated base and during this time the flourophore is excited and fluorescence detected 6. flouorphore attached to p so cleaved and next nt added.

Answer 177

- can detect methylation as the pol pauses for slightly longer on modified bases - circularised molecule so it can b read multiple times to produce a consensus seq and remove random errors (up to 15%) - very fast - but limited input due to number of ZMWs that can be read per SMRT cell - expensive

Answer 178

sequencing by synthesis. The DNA pol is tagged by a donor flourophore, which when bough into close proximity with an acceptor flourophore results in a FRET signal. After incorporation the flourophore attached to the nt is released as it is bound to the phosphate group 1. DNA is covalently attached to the coverslip of a TIRF based micrscope 2. unviersal primers are added to the gDNA 3. labelled pol added to slide because the DNA pol is not attached to a solid substrate it can be exchanged mid sequence - therefore damaged pols can be replaced enabling a high read length

Answer 179

ssDNA is electrophoretically driven through a nanopore as linear denatured DNA - an eletrical potential is applied to a solution either side of the pore - DNA passing through the pore results in a measurable change in the ionic current - due to the lenght of ht pore the change in the current is due to a string of bases called a k-mer not a single ni

Answer 180

Biological nanpore - TM protein channles in a substrate (lipid bilayer, liposomes) - this gives a highly reproducible nanopore size ans structure

Answer 181

Minion is a consumer nanpore sequncer, it is inexpensive, portable, hand held device that can sequence upt to 200kb - DNA to be sequenced is linked to an unzip enzyme so it is thread through the pore as ssDNA. The DNA is also altered so their is a hairpin structure at one end so both strands are thread through, 1 after the other. this provides a continuous read whihc is re-aligned to form a 2D consensus sequence

Answer 182

- fast - portable - as Kmers are measured there are 1000's of possible signals rather than just 4 therefore there is a high error rate especially for indels and homopolymers longer than the kmer lenght - has been used to genotype salmonella outbreak in a UK hospital in 6hrs, seq Ebola in the field (monitor transmission history and viral evolution)

Answer 183

made of graphite with drilled pores and as DNA is passed through the electrical signal reflects the size and confirmation of the DNA molecule in the pore. More stable than biological nanopores and more control over pore diameter, channel length - lower sensitivity to external parameters e.g. pH, temp, salt conc,

Answer 184

DNA is fragmented into 10kbp molecules of DNA, clonally amplified and barcoded before sequencing with a short read instrument - long reads are then synthetically generated from short read sequences very high accuracy (>99.9%) but read lengths are shorter than ohter 3rd generation tech as requires long range amplification and long reads are synthetically generated prone to bias in regions with high GC content or tandem repeats

Answer 185

similar to illumina but uses oilbased emulsion and multiple displacement amplification to ligate short barcoded sequences across a longer molecule unlke illumina it does not aim to generate gapless coverage from short reads, but provides gapless coverage by ensuring there are multiple long fragments originating from the same region in the library prep

Answer 186

* Time- Most of the current systems do not generate enough data fast enough for a rapid response eg in a hospital setting for monitoring infections. * Storage and bioinformatic solutions are required to handle the wealth of data being generated. * Utility and ethics- The vast number of tests and availability of direct to consumer testing raises questions of consumer understanding and personal impact of results, and problems of false positives and negatives.

Answer 187

- mode of inheritance - mutational mechanism - phenotype, age of onset, penetrance and variabiliy - protein structure (functional elements) - strength of the gene-disease asscociation if a gene is part of a phenotype specific panel for a patient there is a greater prior probability that a variant in one of the genes will be associated with the patients phenotype

Answer 188

- population frequency (gnomad) - quality of the call - mode of inheritance aim is to reduce the number of variants for interpretation

Answer 189

MDT is useful escpecially for rare diseases and NGS variants as it allows the variant in question to be interpreted in the context of the patients phenotype. usually involves clinical geneticist and scientists +/- the referring clinician and experts in the field

Answer 190

``` class 5- 99% probability of being disease causing (actionable) class 4 - 90% (actionable) class 3- VUS class 2- 10% class 1 - 0.1% ```

Answer 191

1. literature search 2. mutation databases 3. in silico predictions 4. splicing 5. functional studies 6. inherited/de novo 7. RNA studies 8. IHC 9. segregation 10. co-occurence with a known pathogenic mutation (different impact for AR and AD) 11. LOH 12. enzyme assays

Answer 192

google, pubmed- use HGVS and legacy nomenclature | some software packages collate the information

Answer 193

population databases e.g. gnomad can rule out population polys decipher, HGMD for variants- be aware of miinterpreted info ~27% of HGMD variants listed as pahtogenic are actually polys disease specific databases e.g. LOVD

Answer 194

for AR varaints can support pathogencity (need to prove variants are in trans- parental studies) for AD variant this can refute the pathogenicity of the variant where 2 could be lethal e.g. BRCA

Answer 195

use jarvik and browning paper to determine the level of evidence that can be assigned - co-segregation with disease in affected family members supports pathogenicity and there is more evidence if it is found in multiple unrelated families- no. of informative meioses can be summed - co-segregation in family members without disease support being benign - need to consider phenocopies, variable penetrance, age of onset, - apparent segregation does not confirm that the variant is pathogenic as it may be in linkage disequilibrium with the pathogenic variant

Answer 196

for AD disorders presence in unaffected families members support benign whereas absence supports pathogenicity. need to consider penetrance e.g. 22q11.2 can be inherited from an unaffected parent but may be pathogenic in a child- consider if if fits the phenotype

Answer 197

make predictions based on: - species conservation- the more variability at a position the less constraint and so more likely the variant will be tolerated. Need good quality alignments from multiple species and gaps in the alignment can affect the result so these should be removed - grantham distance - protein software e.g. alignGVD (combines grantham distance with alignment) or polyphen and SIFT (seq alignment and 3D structure) - CADD score can only be used as supporting evidence PP3

Answer 198

variants affecting +/-1/2 splic consensus sites will affect splicing. Splice predictions can be poor in the rest of the gene. However an affect on splicing should be considered for missense and synonymous variants, especially when they affect AG or GT dinucleotide sequences are formed Splice finder like, MaxENTScan, GeneSplicer RNA studies are required to confirm an affect on splicing

Answer 199

to investigate the affect of splicing on cDNA need to consider: - normal isoforms (may complicate interpretation of results) - expression of mRNA in blood- is the trancript of interest expressed in blood or does another tissue need to be sampled - quality of RNA- degrades quickly - NMD, confirmation of biallelic expression of the variant can rule out NMD. If not not variant isoform may not be present as degraded by NMD.

Answer 200

LOH is important for investigating the pathogenicity of TSGs. If a variant in a TSG is detected in constitutional DNA, LOH of the normal allele in tumour can support the pathogenicity of the variant whereas LOH of the variant refutes this. - LOH studies assume the second hit is a large deletion - presence of normal tissue in tumour sample may obscure result so need precise dissection - If LOH of normal allele seen it does not confirm pathogenicity

Answer 201

can provide in vitro demonstration of molecular consequence e.g. disruption of expression or misfolding or in vivo recapitulation of a human phenotype in a model organism - requires identification of a measurable property associated with fucntion - useful info but not definitive. If the result is negative need to consider if the assay was performed in the appropriate genomic context, organismal context, developmental context and a positive result does not confirm pathogenicity e.g. a mutation may be shown to influence transcriptional regulation but is is difficult to assess the significance of an increase in transcription enzyme assays- useful for disorders that result from enzyme deficiencies e.g. non-ketotic hyperglycinemia due to deficiency in glycine cleavage enzyme IHC- MMR genes, DMD- loss of expression

Answer 202

1. there is a submicroscopic imbalance at a breakpoint 2. results in disruption of a gene 3. gene is separated from gene regulatory elements

Answer 203

- can be investigated by arrayCGH - cannot use FISH unless have a suspicion of the gene involved so cannot be used to investigate novel genes e.g. t(6;8) found to have 8q12 dletion at breakpoint- included CDH7 gene resulting in CHARGE syndrome

Answer 204

GOF- splicing together exons from different genes resulting in a novel fusion gene- common in cancer but rare in inherited disease e.g. BRC-ABL1 in CML t(9;22)(q34;q11)- consituitively active tyr kinase LOF- if a gene falls across a breakpoint this can disrupt expression resulting in haploinsufficiency e.g. X;A translocations - to avoid imbalance there is skewed X inactivation favouring inactivation of the normal X. This can result in expression of an XL phenotype if a gene is interrupted on the translocated X. This has been reported in DMD in a female Kleefstra syndrome found in a pateint with a t(x;9) and found disrupted EHMT1 gene in 9q., Further studies found 3 patients with microdel incl EHMT1 = established that HI forf EHMT1 is sufficient to cause Kleefstra syndrome

Answer 205

1. positional effects - can separate a gene from its regulatory regions e.g. promoter - rearrangement may switch regulatory elements with another gene - changes in chromatin structure - euchroamtic can be move to a region of heterchromatin (active to inactive0 e.g. PAX6 HI causes aniridia. Also described in patients with translocation breakpoints 3' to PAX6

Answer 206

large dels and duos >5-10Mb can be detected by karyotype and smaller for arrray. Now evident that the majority of microdel or dup syndromes are due to the effects of a single gene. come are due to contiguous gene del e.g. PKD and TSC resulting in TSC with early onset chronic kidney disease miller-dieker syndrome- type 1 lissencephaly with facial dysmorphism. LIS1 gene loss has been identified as the cause of lissencephaly

Answer 207

breakpoints can directly interfere with a gene or have a positional effect similar to a translocation

Answer 208

co-occurrence of a balance rearrangement and phenotype may be coincidence and mask the presence of a second undetected genetic abnormality regions can be large and may contain may genes- difficult to decide which to investigate. Can look for other patients with the same phenotype to narrow down the candidate gene contiguous gene deletions or duplications may be due to HI or TS for more than one gene in the region

Answer 209

DDD project for DD- used array CGH and WES | 100K- WES (mainly of trios)

Answer 210

- used for genetically heterogeneous but clinically well define disorders e.g. epilepsy - can be virtual or targeted to specific genes during library prep Example- Hu et al used a paenl targeted to the genes on the X chormosome to investigate the cause of Xlinked ID in males and identified 7 novel genes including CLCN4 consideration: - requires a list of candidate genes - usually close to 100% coverage - requires knowledge of the biological systems involved in disease.

Answer 211

sequencing of all the coding regions of human DNA ~20,00 genes and 1-2% total genome - most commonly used ,ethod - suitable for genetically diverse cases and or multiple patterns of inheritance - no bias from using candidate gene list as all genes are sequenced - cheaper than WGS, lower interpretation burden and less data to store - may miss varaiant from incomplete coverage e.g. GC rich, homologous or repetitive regions - less able to detect SVs than WES - no coverage of non-coding regions (although variant in these regions are still a challenge to interpret)

Answer 212

sequencing of the whole genome including non-coding region - use statistical analysis and bioinformatic filtering to identify likely variants or genes - no bias, coverage of non-coding regions, better coverage of repetitive and GC rich regions, better detection of CNVs and SVs, can detect mosaic variants with sufficient coverage - but huge amount of data generated which is costly to analyse - still has limited coverage of tandem repeats e.g. trp repeats

Answer 213

1. filter out poor quality calls or those with a low minimum read depth (although this may mean that mosaics are missed) 2. filter out population polys (Gnomad)- need to be aware of common mendelian or cancer genes e.g. F508 in CF 3. does it fit with the inheritance pattern seen in the family? 4. predicted consequence- LOF, missense, splice site- variants can be filtered based on this prediction. Usually investigate LOF variants first as they are the most likely to have impact on gene function 5. specialist statistical tests to ID gene association with disease e.g. CAST Cohort allelic sum test ) compares total amount of variation in a gene between cases and controls 6. systems/biological pathway analysis- genes can be filtered for those in known disease related pathways for those that interact with other genes previously associated with a similar phenotype.

Answer 214

AR- sequence siblings to ID variants and parents to confirm. Expect homozygous variants in consanguineous families and compound het in non-consanguineous AD- het variant in affected bu not unaffected family members. Can start with trio and mapping can reduce the number of individuals to test dominant de novo- trio. variant present in proband but not parents Xlinked recessive- test affected male. should not be present in unaffected males but will be in carrier females. The more distantly related 2 males it is found in the greater the probability that it is disease related Mosaic variants- compare variants in affected and unaffected tissue- this is usually sufficient for dn variants. Tissue of interest may not be accessible. Possible in skin e.g. for NF1

Answer 215

genetic- likely pathogenic variant which segregated with disease. experimental- protein interactions with previously implemented gene - has biological function consistent with pheno - expressed in relevant tissue and developmental time - can reproduce phenotype in model organism or rescue phenotype with WT

Answer 216

interpretation - difficult in non-coding regions - functional studies can be complex and expensive or require patient tissue that is not always available cohort size- often very few patients or small families available with rare phenotype - highly accurate phenotyping required to combine cohorts - risk of incidental findings (WES/WGS)

Answer 217

- RNA-seq- functionally validate NGS results - CHIP-seq- DNA protein interactions - availability of genome wide data sets will improve- also need better data sets for non Caucasian populations - improvement in data sharing will faciliate interpretation - better undertansing of complex diseases and late onset/variably penetrant disorders

Answer 218

Precious sample, usually limited material available and irreplaceable - therefore long term cultures are established and are grown in situ. This is in case additional DNA is required for testing or FISH or karyotype analysis is needed for follow-up testing - to increase the success rate and reduce the risk of contamination or culture failure 2-3 cultures should be set-up using 2 different media batches and stored in 2 separate incubators. only 1 culture should be harvested at a time and cultures should not be discarded until the final report has been issued

Answer 219

cytotrophoblast are most distantly related to the fetus than the mesoderm. Short term incubations of CVS (for approximately 24 hours) for cytogenetic analysis reflect “spontaneous” trophoblast cell divisions. In contrast, longer-term cultures of CVS consist of the mesenchymal cells and thus represent the ICM - can follow up abn result on uncutlured CVS with cultured CVS to determine cell lineage and how likely it is to represent the fetus -uncultured CVS if chpeed/digested will contain both cell lineages

Answer 220

needs to be carefully dissected to remove any maternal decidua (microscrope and needles/forceps). Morphology and weight of CVS is estimated and cultures are set-up- any remaining cleaned villi is kept in the fridge until the report has been authorised. CVS is spread over the gowing curface and gorwn in chage media at 37oC - change contains the additives- fetal bovine serum, glutamine for growth, antibiotics to prevent infection and bicarbonate to buffer and control the pH

Answer 221

centrifuged, pelleted and supernatant removed. pellet is resuspended and used for DNA extraction or resuspended in amniomax for culturing (same additives as chang)

Answer 222

cultures are assessed after 6-7 days, if colonies are present the media is replaced. - media is chaged to ensure optimum conditions for growth - If 5+ active colonies present the culture should have enough metaphases for harvesting - to get methaphases you need actively dividing cells and overgrowth a a culture can limit the no. of cells (contact inhibition) - 'full'/confluent cultures can be split and supplementary cultures set-up to provide space and ensure the cultures remain active until the case has been reported

Answer 223

should be carried out in a class 2 hood as uses the mutagen BrDU - BrDU (longer chromosomes) and colcemid (inhibits the mitotic spindle) are added to the cultures the afternoon before harvesting -following morning the media is decanted and the cells are resuspened in trypsin -centrifuge to pellet cells and supernatant is removed - resuspend cells in hypotonic solution so cell swell for optimal chromosome spreading add fix (dropwise at first to prevent cell clumps forming) cells are now biologically inert and can be handled without gloves and slides can be made. Temp and humidity is controlled to produce consistent high quality metaphse spreads- left to dry, aged in oven and checked for metapahses under a phase microscope before staining

Answer 224

used to count cells and allow for appropriate seeding of the culture so the media is not exhausted by overseeding but that there are enough cells cultured for analysis

Answer 225

- usually use BM - due to the aberrant increased growth of acute leukemia's most do not require PHA for stimulation, chronic leukemia's with slower growth may do. neoplastic cells are already dividing but in a normal individual white blood cells are non-dividing and need a mitogen to cause them to de-differentiate back to an immature state often a variety of cultures are set-up to try and capture the mutant - 24hour, stimulated/unstimulated, long term no synchronisation

Answer 226

1. obtain your sample e.g.PB 2. add to culture vessel (tube) with growth media (RPM1640) and stimulants as required 3. synchronise cell cycle to obtain maximum number of metaphase chromosomes 4. arrest in metaphase- add colcemid 5. add a hypotinic solution (KCL) to swell cells so chromosomes are spread and not tangled and tight 6. fix cell suspension (carnoy's fix- add dropwise to start to stop cells clumping) 7. make slides 8. satin for analysis e.g. giemsa staining for standard g-banding

Answer 227

amnio acid, balanced salt solution, Hepes buffer, energy source, hormones, pepetides, fatty acids L-glutamine, antiobiotics fetal calf serum and PHA often added PHA

Answer 228

T stimulate mitosis. It is a mitogen that de-differentiates leucocytes back to an immature state where they can undergo division not routinely added to acute leukemias because stimulation if division may interfere with analysis of spontaneously dividing malignant cells.

Answer 229

synchronised to obtain maximum metaphases unless a rapid result is required. - 72 hour culture process thymiding is added to block DNA synthesis so cells are paused in s-phase. Thymiding inhibtis the uptake of DOC (deoxycitidine) causing DNA synthesis to slow down - Excess DOC is added to remove block, all cells then leave S-phase together and are synchronised

Answer 230

After releasing the S pahse block cells progress through the cell cycle to metaphase ~4.5 hours colcemid is then added which blocks the cells in S-pahse and prevents them progressing to anaphase. The longer the culture is in colcemid the more metaphases will be harvested but they will be shorted so need to balance the two requirements.

Answer 231

Fix removes water from cells, kills and preserves them (biologically inert and can be handled without gloves) and harden the membranes and chromatin - preserves the cells and chromosomes

Answer 232

Trypsin is used to digest chromosomes- need to be careful not to over digest as chromosomes become fuzzy with less clear banding. stained with leishmanns stain ``` euchromatin= active, gene and GC rich, early replicating, stains pale heterochromatin= inactive, AT rich, late replicating, stains dark ```

Answer 233

GC rich regions are present in the promoters, enhancers and regulatory regions of genes. They are more stable than regions that are AT rich and a higher melting temp is required to denature the DNA GC rich regions can form hairpin loops and other secondary structures- these can impede the progress of the DNA pol leading to truncated PCR products

Answer 234

- higher melting temp - DMSO and betaine additives facilitate strand separation - formamide is added to PCR products before being separated by capillary electrophoresis. It helps denature DNA by weakening H bonds between strands -

Answer 235

PCR uses thermocycling for repeated amplification of the target to produce 1000's of copies. At each round each of the new products can be used as a template resulting in exponenetial amp (until amount of substrate in the mixture and pol processivity becomes a limiting factor 1. high temp to denature DNA 2. temp lowered to enable primers to anneal to template 3. temp raised to optimum temp for DNA pol and templates are copied * repeat*

Answer 236

PCR is very sensitive to contamination -need to keep pre and post PCR (very high conc so only a small amount could contaminate other samples) separate - can reduce the conc of the starting material to reduce the affect of contamination - run a blank to check for contamination - positive control can detect whether contamination has inhibited the reaction and helps calibrate the analysis e.g. if it has a known fragment size for sizing PCR

Answer 237

Anti-sense oligonucleotides - ss RNA or DNA ~20bp - can bind to DNA and block access to the translational machinery - used to block production of an aberrant protein - correct splicing mutations - correct aberrations by exon skipping - chemically modified to prevent degredation by nucleases - large molecules so enter the cell via endocytosis and need to be stable enough to survive in the endosome

Answer 238

delivery to target tissue - sustained effect- require readministration fo most applications - difficult to achieve complete inhibition due to the relatively large amount of target mRNA in the cell compared to the conc of the ASO

Answer 239

e. g. to convert a DMD phenotype to BMD by inducing exon skipping to restore the ORF so a full length protein is still produced but with reduced functionality - mutations specific but presence of mutation hotspots in DMD mean same treatment can be applied to multiple patients exon 51 skipping is applicable to most patients (14%) - Eleplirsen approved by FDA in 2018 Golodirsen approved 2019 for exon 53

Answer 240

C>T transition in SMN2 results in aberrant splicing and transcripts lacking exon 7 Blocking or enhancing the binding of proteins to an ESS or ISS can increase the amount of full length protein from SMN2 Nusernisen has FDA approval - block the ISS in intron 7, blocking access of the spliceosome and promotes inclusion of exon 7 in the SMN2 mature transcript- this can make up for the lack of SMN1

Answer 241

IONIS-HTT-rx (roche) allele specific ASO designed to target HTT specific SNPs - 2017 1st clinical trial demonstrated that the treatment was safe for the duration of the study and follow-up, and the treatment produces a reduction of mHTT in the cerebrospinal fluid - further studies required to determine the best timing fro treatment and amount of mutHTT lowering required

Answer 242

siRNAs act by RNAi interference to induce gene inhibition- targeted degredation of RNA - formed by DICER which chops dsRNA into 20-25nt fragments dsRNA can be endogenous or exogenous fro a virus or other source - extensivley used in KO experiments - intrinsic cellular mechanism and sequence specificity can be used to specifically target a gene implicated in disease pathogenesis

Answer 243

delivery is a major obstacle | non-specific off target affects as lack the ability for spatial and temporal control

Answer 244

Clustered regularily interspersed short palindromic repeats RNA guided nuclease to enable RNA targeted cleavage and editing- highly specific, once a cut has been made any nearby DNA with matching sticky ends will be incorporated by endogenous DNA repair mechanisms

Answer 245

- straight forward to design and use - main issue is delivery to target cells - highly cost effective technique

Answer 246

amnioglycoside antiobiotics can be used to allow ribosome to read through a PTC to produce the full length protein - amnioglycosides can lead to read through by binding a tRNA to the nonsense codon and insertion of an a.a. - 10% of human disease due to a PTC level of read through generated is variable depending on nature of the PTC - best suited to diseases where low levels of functional protein are sufficient to restore function

Laboratory techniques Flashcards

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