L-16 functional genomics Flashcards
What do functional genomic experiments describe?
gene functions and interactions
What are the different types of study
Protein/DNA interactions
DNA methylation
Gene expression
Protein-protein interactions
Loss-of-function
What is a microarray?
a laboratory tool that analyses large amounts of genes or proteins simultaneously
What do we measure using microarrays
Hybridisation
Where are the samples to probes located on in microarrays
Sample to probes on array
What probe does cDNA have in expression experiment
complementary to coding sequence of known genes
What probe does protein-bound DNA have in ChIP experiment
Whole genome
What probe does whole genome have in SNP experiment
Known SNPs
Can direct sequencing can substitute for hybridisation
Yes
What probe does whole genome have in methylation experiment
Known CpG islands
What probe does whole genome have in CGH experiment
Whole genome
What is high-throughput sequencing
next generation sequencing
What principle is used in microarray and in RNA sequencing
Hybridisation in microarray Direct sequencing in RNA
Steps involved in microarrays
extracting RNA -> cDNA -> biotin labelled cRNA -> random fragmentations -> fragmented biotin labelled cDNA -> cRNA + GeneCHip hyberdisation -> Wash away non-specific
binders and stain with
streptavidin-phycoerythrin ->
Scan array with laser,
detect fluorescence with
CCD, read image into
computer
What resolution is used in microarray and in RNA sequencing
several 100 bp in microarray single base in RNA
What throughput is used in microarray and in RNA sequencing
high in microarray high RNA
is the whole genome is used in microarray and in RNA sequencing
yes in microarray sometimes in RNA
What level of background noise is there in microarray and in RNA sequencing
high in microarray low in RNA
Are microarray and RNA sequencing capable of map transcription and DGE
Yes microarray Yes in RNA
What is the dynamic range in microarray and RNA seq.
Up to a few hundredfold in microarray and >8000 fold
Comapre distinguishng of isoform in microarray and RNA seq
Limited in microarray and yes in RNA seq
Comapre distinguishng of alleic expression in microarray and RNA seq
Limited in microarray and yes in RNA seq
Comapre amount of RNA amount required in microarray and RNA seq
high in microarray and low in RNA seq
Compare cost for large genomesin microarray and RNA seq
high in microarray and relatively low in RNA seq
Which company is dominant in terms of high throughput suquencing
Illumina
What are the grounds for competitionin high throughput seuqncing
driven down cost, increased throughput
Illumina Sequencing
fragments of DNA (library) bound to solid surface (flow cells) -> bridge PCR = clonal clusers -> sequencing in cucles -> modified nucelotides w fluorescent group blocked extentiion = reversible termination
How do we sequence RNA
High througput sequencing technologies to get info about RNA content -> mRNA -> cDNA -> used for sequnecing library gen. -> allws quantification, profiling and discovery of RNA
How does RNA-seq work?
Sequences in final library are derived from RNA population in
sample
* Presence is proportional to original sample
* More abundant RNA species will be present more frequently in library
* ‘Random’ priming is attempt to remove bias
* Or not introduce it
* Actual randomness debatable
What are RNA-seq considerations
Big data sets require expert processing
* Expression data can be noisy
* Careful experimental design important
* Easy for confounding factors to dominate
* Good practise same as for any statistical approach
What are other functional genomics approaches
Big data sets require expert processing
* Expression data can be noisy
* Careful experimental design important
* Easy for confounding factors to dominate
* Good practise same as for any statistical approach
WHat is ChIP-Seq
Cross-link proteins to DNA?
What occurs in ChIP-Seq
Isolate DNA and shear
* Sonication for ‘random’ shearing
* Immunoprecipitate protein of
interest
* Reverse cross-linking
* Purify DNA
* Sequence
What is ATAC-Seq
Assay for Transposase-Accessible Chromatin
What is ATAC-seq similar to
Similar to older DNAse-Seq
* Relies on transposase Tn5
* High activity transposase
* Highly efficient cutting of exposed DNA
* Ligation of adapters to ends
* Adapter ligated fragments isolated, amplified and sequenced
What is bisulphite sequencing
Bisulphite treatment is used to determine the methylation state of
DNA
* Methylated cytosine protected from deamination
* Unmethylated cytosine converted to thymine (via uracil)
What occurs in bisulphite sequencing
Sequencing of bisulphite treated, along with untreated samples
identifies sites of methylation
* High depth sequencing can provide quantitative estimates of
methylation
* Hypo- and hyper-methylated regions can then be identified
(hyper = txn. silencing)
Reduced Representation bisulphite sequencing (RRBS) - what causes this?
Human genome has 28m CpG sites and Assaying all of these in a single experiment problematic
What enzyme does RRBS utilise to enrich for CpG?
Msp1
What does Msp1 result in?
fragments which begin/end with CpG
How much of the genome needs to be sequenced for RRBS?
1%
High throughput sequencing produces ______ amounts of data
large
Data can be ____ and complex
Noisy
Computational approaches needed to make most of data for both
processing and analysis
