MTGE 122

Final Review

DNA amplification

DNA amplification

thermocycler program uses two denaturation stages one at 94C and one at 90 to save time

De Novo Sequencing

Used to generate a DNA sequence for which no prior info is know

Resequencing

Sequencing of DNA followed by the comparison to a reference sequence

Used in clinical diagnosis

2 Steps in PCR product purification for sequencing

1. remove PCR primers

2. Remove dNTPs

Enzyme digest - ExoSAP for PCR purification

Exonuclease - destroys primers

Shrimp alkaline phosphatase hydrolyzes dNTPs to remove the phosphates so they cannot bind to other dNTPs.

Column PCR product purification

Silica gel membrane in column binds the DNA that is the correct length and everything else flows through

Fluorescent DNA sequencing

separation of fluorescently labelled, extended fragments of DNA using capillary electrophoresis for the determination of sequence

Components of enzyme digest in PCR purification for sequencing

1. Exonuclease to destroy primers

2. Shrimp alkaline phosphatase (SAP) hydrolyzes dNTPs so can’t bind

Column PCR product purification for sequencing

Silica gel column binds the DNA in the range desired and the primers and impurities flow through

Bi-Directional Sequencing

higher accuracy

1. confirm variants results are accurate,

2. identifies secondary structures

Components of the terminator ready reaction mix 2 things

Nucleotides with diTP instead of dGTP which prevents base compression

Each ddNTP has a dye that emits a unique wavelength when excited by light

Primer Design Sanger - PCR primer with Sequencing tail

M13 is added to the 5’ end

allows for single MM in the FWD and RVS reactions

Primer Design Sanger - PCR primer is the Sequencing primer

use a PCR primer as the sequencing primer

• can result in excess pipetting

Primer Design Sanger - Nest sequencing primer

primer binds within the PCR product (eliminates non specifics)

Sequencing product purification - Gel Column

Sephadex spheres separate products based on weight - the leftover dyes are caught in the pores

• fast but dye blobs present

Sequencing product purification - BigDye Xterminator

Solution has an insoluble phase to capture the ddNTPs and stabilize the sample

• requires special vortex

Sequencing product purification - Ethanol

precipitation with EDTA and ethanol then incubate and wash

• slow

Capillary Electrophoresis Settings for Sanger

50 cm Capillary - separates up to 800 bp of sequence

POP7 - intermediate polymer

Spectral Calibration with Z dye

Minimal overlap, no dips or irregularities

peaks are separate and distinct

ordered

Blue, green, yellow red

3 rules of nucleotide number in HGVS

nucleotide 1 is the A of ATG

5’ of the A is -

3’ of the stop codon is *

Intron naming in HGVS

• total nucleotide divided in half

• nt in first half are numbered in reference to the last exon

• nt in the second half are numbered in relation to the downstream exon

3 keys points about RNA

• contains ribose

• uracil instead of thymine

• RNA has OH at carbon position 2

RNA isolation methods - Trizol and Phenol

• lyse cellular and nuclear membranes

• add chloroform

• centrifuge

• precipitate RNA with EtOH

RNA isolation methods - RNA kits

Column Chromatography uses silica membrane with high salt to bind the big RNAs and then can be eluted

consider - column can clog and DNA must be sheared prior to loading

Ideal Quantitation values for RNA

absorbance at 260nm

260/280 - 1.8-2.1

260/230 - 2.0

Ideal quality bands for RNA

Quality

• 28S band at 4.7kb

• 18S band at 1.9kb

rRNA

many sizes - form the large subunit of ribosome while associating with other proteins

tRNA

inverted four leaf clover shape with anticodon in the bottom loop - carry AA to ribosome

hnRNA

heterogeneous RNA rapidly degraded

snRNA

small nuclear ribonucleoprotein only in the nucleus and splice the hnRNA

mRNA

100-10,000bp

found in cytoplasm and is translated into proteins

siRNA

dsRNA

small interfering RNA silences genes

Uses of QF-PCR

Rapid aneuploidy detection SCREEN for prenatal diagnosis - must be confirmed with ultrasound findings to be reported

screens for 13,18,21,X/Y

What does qf-pcr amplify

Amplifies polymorphic markers called short tandem repeats which can be separated by fragment analysis to detect copy number

Short tandem Repeats qfpcr

Polymorphic loci of 2-7nt repeats that are inherited in different numbers from each parents

• the allele inherited from the mother and father at one locus are different creating unique genetic fingerprint

7 reasons microsatellites are used for analysis

1. Present in large numbers in the genome

2. Evenly spaced in genome

3. Alleles are inherited in a Mendelian manner

4. Highly polymorphic

5. Stable over multiple generations

6. Can be specific to certain populations

7. Size is Ideal for amplification (even in degraded samples)

Prenatal sample requirements for qfPCR

Optimized to work on low levels of DNA 1-10ng

• 1ml Amniotic fluid

• 0.2mg chorionic villus

• Fetal blood (5ul)

Data interpretation of qfPCR

Peak areas of the different alleles for a single marker are compared to generate a ratio

• amount of product is proportional to the amount of original target

Peak areas of the two alleles on a single marker are compared to each other to generate a ratio

PA1/PA2=ratio

ISCN for qfPCR

“rsa” tells region specific assay

rsa(X,13,18,21) x copy number

Uses of Microarray

investigate copy number changes throughout the entire genome using hybridization of patient DNA to probes on a slide with a detection system

2 Clinical applications microarray

1. Copy number changes

2. Genotype - SNP analysis

miccro array chip

solid substrate with assay material on it - used for single channel

Agilent with OGT platform manufacturing

uses an inkjet printer to place nucleic acid onto a glass substrate creating 60-mer oligo probes either depositing pre-synthesized cDNA or synthesizing base by base

Microarray - probe

series of nt for a complementary unique base pair with the genome are affected to the glass of the microarray

Microarray - feature

A large grouping of the same probe for a locus

Found around the array with each features representing a different area of the genome

Whole Genome Microarray

Equally distribute probes provide coverage of the whole genome

detects aneuploidies and large imbalances

target only Microarray

targets clinically relevant regions with high density of probes to ensure genes are well covered

detects small changes at the relevant gene

Targeted whole genome Microarray

provides coverage of the entire genome with increase coverage of target regions

CGH and SNP Microarray

Most clinically relevant

provides whole genome coverage with targeted coverage and loss of heterozygosity information via SNP probes

3 Consideration in array resolution

1. probe size

2. number of features

3. distance between probed loci

Probe Size in microarray resolution

smaller length = more specificity =high resolution

longer = lower resolution

Number of features in microarray resolution

Greater number of features = higher resolution

Distance between probes in microarray resolution

Smaller distance between loci = higher resolution

Quality Control Metrics for Microarray

1. DLR spread

2. red/green signal intensity

3. background noise

4. Signal to noise ratio

5. Reproducibility

6. array image

Microarray analysis - DLR spread

Measure the difference in Log 2 ratio between all pairs of probes on a chromosomes and expect them to be aligned

If DLR spread is too high it indicate variability between neighbouring results and makes it hard to call abnormalities

Microarray SNP analysis

SNP analysis gives insight into the genotype status of the patient to detect Loss of heterozygosity and mosaicism

SNP probe algorithm - microarray

only the DNA fluorescence is measure giving an intensity representative of how much alleles is bound - genotype is inferred by the comparing the intensity of the two features for each SNP (A vs B)

B alleles frequency plot

graph representing the presence of each genotype

can detect mosaicism at low levels 10-15%

How to calculate consanguinity

Add autosomal LOH segments >5Mb

Divide by size of autosomal genome

convert to percentage

Depth of coverage

how many times a base has been sequenced or read - a greater depth of coverage increases detection sensitivity and reliability of results

3 considerations for library prep of NGS

1. not using EDTA and chelating salt - inhibit enzymes

2. extract into EB, low TE or water

3. store at -20 do not freeze/thaw

2 types of fragmentation in NGS

Physical shearing

enzymatic digestion

Physical shearing in NGS

break the bond of DNA using focused acoustic, ultrasonic energy on the sample within the Covaris for unbiased fragmentation

Enzymatic Digestion fragmentation in NGS

restriction endonucleases break both strands however where may be recognition site present leading to biases

4 steps in library prep for NGS

1. fragmentation

2. end repair

3. A tailing

4. adapter ligation

2 types of target enrichment in NGS

1. Hybrid capture

2. Amplicon based capture

5 steps in Hybrid capture for NGS

1. denature DNA

2. hybridize biotin to the targets

3. Capture target on bead

4. recover target

5. amplify

4 steps in Amplicon based capture in NGS

1. primer based PCR amplified regions of interest

2. produces amplicons

3. PCR and barcode adapter ligation with second PCR

4. Data of reads aligned to the reference

Size selection in NGS

fragments outside the desired range interfere with sequencing so should be removed by purification

3 methods of purification for NGS library

1. gel electrophoresis

2. bead based purification

3. double sided bead clean up

Double Sided bead clean up for NGS

1. lower ratio bead:DNA captures large fragments

2. wash

3. higher ratio bead:DNA captures wanted DNA leaves small fragments

4. Wash

Quality control steps in NGS

Bioanalyzer does automated electrophoresis to quantify library and asses fragment sizes

Sample Pooling considerations in NGS

all libraries can be pooled together if there are in the SAME size range and concentration

4 steps to load the NGS library

1. quantified by quantitative PCR

2. internal control added

3. denature with NaOH or heat and ice

4. load with reagents and buffer onto the flow cell

4 Channel base calling

4 channel requires 4 images after each cycles to capture a unique dye for each base

2 Channel base calling

Taking only 2 images after each cycles and can determine all 4 bases using computation

Pair end sequencing

After the first sequencing by synthesis the strand folds over hybridizes and DNA pol extends the reverse strand

5 benefits of paired end sequencing

1. more accurate

2. precise mapping

3. detect indels

4. remove duplicate artefacts

5. higher number of single nucleotide variation

Optimal read length

Median insert size should be 2x longer than read length so only a few bases overlap

Primary data analysis NGS

algorithm generates a base call with a quality metric

data is pooled and demultiplexed

Secondary Data analysis NGS

Read alignment by mapping position sequence to the reference and creates a map

variant calling programs determines single and multiple base pair variation

Tertiary Data analysis

Genetic variation put in a list with chromosome number, position and reference alleles

Ion Torrent Platform

Clonal amplification where DNA fragments are mixed into emulsion oil and beads, DNA fragments achor and are amplified

Clustering Quality

Cluster density

Cluster passing filter

Cluster density

how dense the clusters are on the flow cell expressed as cluster per mm

Cluster passing filter

measure of the number of clusters used for final data output, uses a digital filter to remove low quality clusters

Q score

measures sequencing accuracy by indicating the probability that a base is called incorrectly across the entire run and in each variant

Alignment process in secondary analysis

1. Input the patient generated file and reference genome

2. alignment platform/program

3. output as a binary alignment map

Depth of coverage NGS

Average number of times a base is sequenced

Breadth and Uniformity in NGS coverage

All regions have even coverage and are on target

Variant caller for secondary analysis

Determine where difference between the patient and reference exist and annotate

Background on BRCA1

Tumour suppressor gene involved in DNA repair 17q21.31

when inactive allows accumulation of defect causing cells to divide

Multiplex Ligation-Dependent probe amplification

relative method to detect copy number variations from one PCR based reaction

target gDNA quantity for MLPA

50-100ng must be accurate

parts of SALSA MLPA probes

1. PCR primer

2. stuffer sequence

3. hybridization sequence

Purpose of Q fragments for MLPA

Confirms quantity of DNA sample is sufficient

Ligation and DNA independent

Purpose of D fragments for MLPA

Determines adequate sample DNA denaturation

Ligation and DNA dependent

Benchmark/ligation fragment

used as a benchmark to compare the other CQ fragments and confirms the probe to target hybridization and ligation are complete

Ligation and DNA dependent

MLPA detection of known point mutations

probes designed for certain SNPs and only perfectly matched probes are ligated/amplified