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114 Cards in this Set
- Front
- Back
- 3rd side (hint)
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What is functional genomics?
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The study of how things with different genotypes can have different functions, i.e. phenotypes!
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How big the genome?
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3Gb. 6 meters in each cell!
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What different types of proteins are there.
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Structural, regulatory__Housekeeping (general), Luxury (specific)
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Why might RNA not be a good proxy for proteins?
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asf |
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What are the 4 analyses you can do with RNA-seq?
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Expression levels, DE, patterns in expression, splicing and isoforms
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What is cDNA?
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DNA strands produced from RNA via revere transcriptase.
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Name 2 ways to measure RNA
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Microarrays__RNA-seq
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Explain how microarrays work
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Probes with solid support.__Hybridise labeled samples of mRNA to these.__Shoot with lasers.
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Explain how RNA-seq works
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asd |
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Name elements in transcriptional regulation
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Gene/protein interactions__DNA methylation__Chromatin (epigenetic structure)__microRNA__Gene expression
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Explain 2-color microarrays
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Experimental culture (red) + control culture (green)__Measure colour
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Explain 1-color microarrays and give examples of types
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BeadArray: Beads coated with copies of probe__23 base address linked to 50-base gene specific probe__30 copies of each bead type per array: 47k genes => 1.3M probes
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Explain the general workflow when working with microarrays
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asf |
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Explain the three cornerstones of experimental design
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asd |
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How to detect outliers?
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log-2 normalisation__Positive/negative controls__Number of deteceted probes per sample__Signal distribution__Clustering / PCA
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Explain how control probes work
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Positive: Houskeeping genes -- should be there!__Negative: Things that should not be there!__Negative also used to determine background.
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What do MA-plots show?
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log2 fraction vs. mean of logs. Should be straight.
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Name some normalisation techniques
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Simple scaling, LOESS, Quantile, robust spline
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How does quantile normalisation work?
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asf |
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What can be done to determine normalisation?
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MA-plot / PCA__Search for patterns in data
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Describe the RNA-seq workflow
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Align to sequence (genome/transcriptome), get annotation, compare with databases
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Describe why and how to filter probes
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p = 0.01 => lots of finds! 50k probes -> 500 false pos.__Remove probes not present in all samples.__Use multiple probes per gene
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Describe the three downstream analsysis categories
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Class discovery: clustering, PCA__Class comparison: groups known, genes / pathways associated?__Class prediction: SVM, survival
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What is the cross-hybridisation problem and what does it limit?
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In microarrays, dna tags to the wrong probes.__Sensitivity, range, probe coverage
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Benefits of RNA-seq
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Can get isoform data -- not possible with microarray__Gives absolute abundance
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Benefit of microarrays
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Faster, easier, more mature analysis
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How does sequencing work?
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Reversible terminators__Sequencing by ligation__Nanopores
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Describe the Solexa sequencing structure
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15 steps or so
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What are some modes of sequencing?
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Paired-end: each end aligned separately__Multiplexing__Capture sequencing
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What are some benefits of paired end sequencing?
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Disambiguate non-uniquely mapped reads__Detect isoforms__Detect duplications, inversions, chromosomal rearrangements__Calculation of distribution of insert sizes
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Describe multiplexing (probably not in it)
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Look it up
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Describe capture sequncing
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Magnetic strip to specific sequences__Flush away rest.
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Discuss problems with sequence alignment
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Filtered / unfiltered (???)__Unique / non-unique__Duplicates (amplification bias)__Mismatches / indels__Adapters and index sequence
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Sequencing trends (probably not)
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sa |
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What questions are RNA and ChIP seq answering?
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RNA: 1) WHAT is the sequence? 2) WHAT is he concentration?__ChIP: 1) WHERE does it bind? 2) HOW MUCH is bound?
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Why is RNA-seq imperfect?
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Data consists of 1-2 sequences per fragment__Base call qualities for each base in each read varies__RNA-data is meta-data. Read -> cDNA library__Reference genome rarely sample genome, SNPs etc, indels, structural variants__Reads prone to error (1/1000)
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Describe the ChIP-seq protocol
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1. Crosslink and shear__2. Add protein specific antibody and immunoprecipitate__3. Sequence one end of each fragment__4. Get coverage!
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Describe the RNA-seq protocol
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1. Select RNA of interest (e.g. mRNA)__2. Fragment and reverse-transcribe to dsDNA__3. Size select, denature to ss-cDNA__4. Sequence n bases from one/both ends of fragments (n ~ 50-100)
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How to map RNA reads to transcripts?
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De novo assembly(???)__Alignment + gene model assembly (map to DNA)__Transcriptome alignment (map to RNA)
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Explain how de Bruijn graphs work (VERY POSSIBLE EXAM QUESTION)
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asf |
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What is a kmer?
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A substring of a read
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Explain how to choose k in RNA-seq assembly
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asf |
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How do SNPs/errors appear in Bruijn graphs?
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Bubbles! Take path with highest coverage.
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Explain differences between genome and transcriptome alignment
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G: Detection of novel genes. Spliced alignment is tricky. Insert sizes harder to interpret.__T: No need for spliced alignment. Simplifies read counting for each isoform. Simplifies discrimination between mappings using insert sizes. Novel genes go undetected.
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What is TopHat? Describe workflow.
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RNA-seq aligner.
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What is a gene model?
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asf |
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How does Cufflinks work?
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asf |
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RNA-Skim, kallisto, Sailfish -- hash-table aligners
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asf |
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Describe briefly how to filter alignments
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Pick part SNP / variant with best coverage__Multiply matching sequences with outlying insert lengths__Take out repeats (sole peaks)
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Why are isoforms interesting?
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They give increased resolution of RNA-sequence (the variants)__We have two versions of each isoform sequence in diploid orgs
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What is a Poisson distribution?
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Independent events occuring at given rate__Mean=Var=r8, pets_r8=dogs_r8+cats_r8
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What are three determining factor for how many reads align to a transcript?
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Total nr reads__Length of transcript__Abundance of transcript
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What is a formula for estimated gene reads?
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r_g = Poisson(b mu_g l_g)__mu_g: concentration, l_g effective length, b: norm
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What are some problems with Poisson models?
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Gene length ambigous due to isoforms__Cross-linkage__We don't get reads of isoforms
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Explain the multinomial distribution
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>2 categories. Good when there are multiple SNPs and isoforms.
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MMSEQ
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asf |
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Transcript amalgation
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asf |
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What is gene imprinting?
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Genes are expressed in a parent-of-origin specific manner__Gene from father imprinted -- only mother version expressed
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What are the aims of read count normalisation?
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Comparable across features (e.g. genes)__Comparable across samples__Human-friendly scale
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What is RPKM normalisation?
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Set k_ig such that estimates of mu_ig are comparable between genes and samples.__muhat_ig = r_ig / k_ig
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From Binomial to Poisson
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awff |
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TMM normalisation
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asf |
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Median log deviation normalisation
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asf |
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What is a negative binomial distribution? When do we use it?
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When rate of Poisson dist not fixed, but varies according to a gamma dist.__Variance is greater than mean.
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How does ChIP-seq work?
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1.Isolate chromatin 2. Cross-link and fragment 3. Antibodies, precipitate 4.__Reverse cross-links, purify DNA 5. Ligate adaports, sequence
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What is ChIP-seq interested in?
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Where is the protein bound? 90 % background!__Where are proteins bound differentially?__What do the sites mean biologically?
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Describe the ChIP-seq workflow
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asf |
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Why use a control track in ChIP-seq?
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Get background. See tissue anomalies. Open chromatin???. Experimental / technical biases. Background distribution irregular.
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Name three types of controls in ChIP-seq
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Input__Vehicle__Non-specific antibody
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Explain good experimental design in ChIP-seq
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Technical replicates: multiple lanes per flowcell__Biological: patient samples, model organisms__Experimental: repeat procedures, (different antibody)
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Name the important ChIP-seq parameters in expermiental design
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Single end / paired end, read length (50bp), read depth (20-30M), batches and randomisation, multiplexing (one pool is optimal)
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What are blacklists for?
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Duplicates etc.
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How would you perform quality assesment in ChIP-seq?
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Coverage histogram__Fragment length estimation (cross-correlation, cross coverage, normalised__score)
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How do you call peaks?
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Identify maxima. Take region around it.
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Name three peak-based metrics
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1. Reads in peaks (reads that overlap peaks, dist of read density)__2. Peak profiles (mean density at each position relative to summit)__3. Clustering and PCA
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What are the two types of differential binding analysis?
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Overlap: peak/site occupancy__Quantitative: binding _affinity___Binding site count density__Binding profile__Sliding windows
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What are consensus peaks?
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Peaks found in several samples from the same tissue/experiment
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Explain the DiffBind workflow
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1. Read in peaksets__2. Determine occupancy__3. Count reads__4. DBA__5. Plot and report, then re-evaluate.
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Name plotting tools used in DBA
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MA plots__Heatmaps / Clustering / PCA__Boxplots. Peak abundance / density.
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Name as man yas you can of the 7 regulatory elements:
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TFs, Histone mods, Nucleosome pos, chromatin domains, Polycomb group (PcG) proteins, DNA methylation, non-coding RNA.
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Name four types of TFs
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Master regulators, General TFs, Pioneer factors, Tissue specific factors
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What are the three classes of regulatory elements?
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Core promotes__Enhancers & super enhancers__Locus control regions__Silencers__Insulators
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Core promoters
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asf |
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Enhancers & super enhancers
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asf |
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Locus control regions
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asf |
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Silencers & insulators
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asf |
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How to identify regulatory elements?
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Motif analysis__Sequence binding sites
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MEME
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MEME-ChIP
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HOMER
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What is the difference between epigenetics and epigenomics?
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genetics: gene expression__genomics: overall chromatin state of full genome; organism has single genome, many epigenomes
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What is a histone?
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What the DNA coils around -> gives chromatin
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How can histone be modified?
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5 common ways -- see slide
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What are chromatin marks?
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af |
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Chromatin segmentation
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asf |
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WHAT ARE CHROMATIN MARKS?
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asf |
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Explain how Hi-C works
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asf |
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What are some Hi-C technical and biological biases?
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awf |
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A-B compartments
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af |
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How to detect chromatin accessibility?
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awf |
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ATAC-seq
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asf |
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What is a pathway?
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Series of conseq interactions that give rise to a certain state
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What are the three types of pathways?
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Signalling__Genetic / transcriptional / regulatory__Metabolic (ana / cata / energy transport)
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How would you determine enrichment of gene lists?
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Fisher's / hypergeom__Chi**2__Binomial
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How would you determine enrichment of ranked lists?
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Kolmogorov-Smirnov__Minimum hypergenometric test__Wilcoxon rank sum
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Where do the gene lists in PWA come from?
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omics data: RNA / ChIP / proteomics
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What is Gene Ontology?
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Contains information on cellular component, molecular function, biological__process
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What are some causes of SNPs?
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Replication errors__Repair error__Mutagens__Spontaneous
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What are some causes of INDELs?
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Strand slippage__Aberrant repair__Retrotransposons
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What are some causes of Structural Variations?
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Replication errors__Retrotransposition__Repair errors__Recombination errors
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What is a somatic variation?
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Mutations acquired post conception__SNVs, not SNPs__CNAs, not CNVs__INDELs__SVs
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What is the difference between SNVs/SNPs and CNAs/CNVs?
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???
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Explain the copy number calling workflow
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Quantify signal (depth / intensity)__Segment chromosome (HMM / smoothing)__Call changes (threshold, cluster, prob. models)
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B-allele frequency
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af |
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BAF-banding
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fass |
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Some cancer types are mutation driven, some CNA driven
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as |
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