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Genomics Lecture 4

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winniesmith2's version from 2017-10-24 17:00

Section 1

Question Answer
What are microarray sets of miniaturized chemical reaction areas that may be used to test DNA fragments, antibodies, or proteins. General name to analyse many markers together in miniature format.
Describe microarray reactions Each reaction area or spot is having immobilised target (fixed co-ordinates) which is hybridised with complimentary probe present in the testing sample (change at co-ordinates)
How does a microarray workBy complementary binding. Probes on surface; glass beads, chips, slides. High throughput.
What can arrays detect(almost everything) -mRNA (gene expression) -microRNA -methylation -repeat units -SNP
What does throughput mean the maximum rate of production or the maximum rate at which something can be processed.
What is gene expression profiling - Monitoring expression levels for thousands of genes simultaneously
What is Array CGH (comparative genomic hybridization)- Assessing genome content in different cells or closely related organisms
What is SNP array (Single nucleotide polymorphism)- Identifying single nucleotide polymorphism among alleles within or between populations
What is ChIP-on-chip (Chromatin immunoprecipitation) - Determining protein binding site occupancy throughout the genome
What is Methylation arrays (immonoprecipitate methylated DNA)-Determining which regions of DNA are methylated to determine epigenetics
What is a DNA microarray a collection of microscopic DNA spots/patches attached to a solid surface (slide or bead)
What is a probeprobe is used for the nucleic acids of known sequence, which will be attached to the surface in the case of the microarray. Each DNA spot/patch is specific (specified nucleotide sequence) and is termed probe* (or oligo). Each DNA spot/patch contains 10-12M amounts of DNA.
What is target DNA the unknown sequence or collection of sequences to be analyzed.
How does DNA microarray work Probe Hybridisation with target DNA under stringent conditions. Probe-target hybridization detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labelled targets. Determine relative abundance of nucleic acid sequences in the target.
Step by step, DNA microarray testing Sample of target DNA is unzipped and broken into pieces --> DNA pieces are chemically modified and tagged with fluorescent Green Dye AND thousands of standard test genes tagged with fluorescent red dye --> Microarray chip and hybridized DNA --> Analysis of chip under laser light.
Steps of SNP microarray Target prep (Amplify-->fragment). Hybridization (capture+label)(sample binds to array). Ligation (differentiate)(Labelled probes bind to sample, differentiating between the two alleles). Signal Amplification (stain and image)(Make it bright enough then measure intensity of array).
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Section 2

Question Answer
Factors to consider in designing microarray experiments. 1) Need to do lots of control experiments-validate method. 2) Do replicate spotting, replicate chips, and reverse labeling for custom spotted chips. 3) Do pilot studies before doing “mega chip” experiments. 4) Don’t design experiment without replication. 5) Design simple (one-two factor) experiments, i.e. treatment vs. untreatment. 6) Understand measurement errors. 7) In designing databases; they are useful ONLY if quality of data is assured. 8) Involve statistical colleagues in the design stages of your studies.
Challenges in analyzing microarray data. (1)- Amount of DNA in spot is not consistent - Spot contamination - cDNA may not be proportional to that in the tissue - Low hybridization quality -Measurement errors -Spliced variants
Challenges in analyzing microarray data. (2) data-Outliers -Data are high-dimensional “multi-variant” -Biological signal may be subtle, complex, non linear, and buried in a cloud of noise -Normalization -Comparison across multiple arrays, time points, tissues, treatments -How do you reveal biological relationships among genes? -How do you distinguish real effect from artefact?
What does P stand for Pixel intensity
What does F stand for feature intensity
What does B stand for background intensity
What does Rp stand forratio of pixel intensities
What does Rm stand forratio of means
What does mR stand for median of ratios
What does rR stand forregression ratio
History of SNP microarray lecture 4 page 15
Scientific supplier related lecture 4 page 16
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Section 3

Question Answer
What is used for pre-processing microarray data analysisNormalisation and scatter plots
What inferential statistics is used for microarray data analysist-test, ANOVA
What is descriptive statistics Distance, clustering, principle components analysis
Genomic sequencing Genome--> Short fragments of DNA --> Short DNA sequences --> Sequenced genome
What is sanger sequencing (chain termination) used forFinding exact ordering of Bases or DNA alphabet. Uses 4 different colours of fluorescent dyes and dideoxy nucleotides. Sequence of nucleotides can be easily typed. Allows comparisons between individuals and populations. Was costly --> now affordable at different levels. Finest level of variation/analysis.
How does sanger sequencing reaction happenits like PCR except: Uses only a single primer and polymerase to make new ssDNA pieces. Includes regular nucleotides (A, C, G, T) for extension, but also includes dideoxy nucleotides ( for chain termination).
Describe steps in sanger sequencing reactions (that differ from PCR) 1) Extension produces a series of ddNTP terminated products each one base different in length. 2) Each ddNTP is labeled with a different colour fluorescent dye. Each different sized fragment runs differently through capillary electrophoresis. 4) Sequence is read by noting peak color in chromatogram (posessing single base resolution).
Sanger sequencing reactions in more detail . A primer that has been designed to recognize a specific region of a DNA template anneals and is extended with a polymerase. Because a mixture of dNTPs and ddNTPs exist for each of the four possible nucleotides, some of the extension products are halted by incorporation of a ddNTP while other molecules continue to be extended. Each ddNTP is labeled with a different dye that enables each extension product to be distinguished by color. A size-based separation of the extension products permits the DNA sequence to be read provided that sufficient resolution is present to clearly see each base.
What is the output of sanger sequencing reactiona chromatogram usually 700bp
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Section 4

Question Answer
What does next-generation sequencing refer to non-sanger-based high-throughput DNA sequencing technologies. Millions or billions of DNA strands can be sequenced in parallel. Removes fragment-cloning methods that are often used in Sanger sequencing of genomes. -2nd generation sequencing, massively parallel sequencing and deep sequencing.
Examples of 2nd generation sequencing -Ion semiconductor sequencing (Ion Torrent) -Sequencing by synthesis (Illumina) -Single molecule real time (Pacific Bioscience) -Nanopore sequencing (Oxford Nanopore) -Qiagen GeneReader (Qiagen)
Commonalities in NGS 1) All NGS platforms are library dependent: -Libraries are generated by amplification or ligation to custom linkers. 2) Each library fragment is amplified on a solid surface: -Bead or flat Si-derived surface -Covalently attached adaptors which hybridise with library adaptors. 3) 100,000’s to 100,000,000’s reaction/instrument/experiment: -Massively parallel sequencing. 4) Digital output/read to produce direct quantification 5)Shorter read lengths than capillary sequencers (700bp v 100-300bp)
What are the 3 steps in massively parallel sequencing1 - Clonal generation 2 - Clonal amplification 3 - Sequencing
What happens in clonal generation-Shear High Mol Wt DNA. -Clean DNA ends (remove terminal overhangs). -Ligate synthetic DNA adaptors (PCR) - Platform-specific adapters are ligated to the fragments. -Produce size fractions (PCR). -Quantitate (to ensure correct dilution profile to enable “clustering”).
What happens in clonal amplification -Amplify library: fragments with flow chamber (PCR) -Denature clusters to ssDNA -Hybridize sequencing primer to linear ssDNA clusters
2 ways library amplified in clonal amplification: fragments with flow chamber (PCR)1) Emulsion PCR – oil-in-water based: One Bead = One Fragment = One Sequence Bead. 2) Bridge Amplification – solid surface, flow-cell based; One Cluster = One Fragment = One Sequence Bead
What happens in sequencing -Technology/Scientific supplier dependent -Sequence alignment and data analysis (GRCh38 – reference human genome). DNA nucleotide sequence (constant output). Detection Chemistry (Variable- technology dependent).
What is prosequencing Sequence incorporation of nucleotides --> luminescence
What is sequencing by Ligation Introduction of oligonucleotide probes-->fluorescence
What is reversible dye terminators Incorporation of reversible dye terminators --> fluorescence
What is ion current variation Measurement of hydrogen atom release-->voltage recording
What is 3rd Generation Sequencing Single molecule sequencing- 0.5-4 hours. 8,500bp. Phospholinked fluorescent nucleotides (seq- by synthesis
What are the effects of next generation sequencing 1) Labour, administration, management, utilities, reagents, and consumables. 2) Sequencing instruments and other large equipment. 3) Informatics activities directly related to sequence production (e.g., laboratory information management systems and initial data processing). 4) Submission of data to a public database. 5) Quality control for sequencing projects. 6) Technology development. 7) Development of bioinformatics/computational tools to improve downstream sequence analysis. 8) Management of individual sequencing projects. 9) Informatics equipment. 10) Data analysis downstream of initial data processing (e.g., sequence assembly, sequence alignments, identifying variants, and interpretation of results).
Reference genome- small printpages 35/36 important
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Section 5