L-16 functional genomics

Question 1

What do functional genomic experiments describe?

Answer 1

gene functions and interactions

Question 2

What are the different types of study

Answer 2

Protein/DNA interactions
DNA methylation
Gene expression
Protein-protein interactions
Loss-of-function

Question 3

What is a microarray?

Answer 3

a laboratory tool that analyses large amounts of genes or proteins simultaneously

Question 4

What do we measure using microarrays

Answer 4

Hybridisation

Question 5

Where are the samples to probes located on in microarrays

Answer 5

Sample to probes on array

Question 6

What probe does cDNA have in expression experiment

Answer 6

complementary to coding sequence of known genes

Question 7

What probe does protein-bound DNA have in ChIP experiment

Answer 7

Whole genome

Question 8

What probe does whole genome have in SNP experiment

Answer 8

Known SNPs

Question 9

Can direct sequencing can substitute for hybridisation

Answer 9

Yes

Question 10

What probe does whole genome have in methylation experiment

Answer 10

Known CpG islands

Question 11

What probe does whole genome have in CGH experiment

Answer 11

Whole genome

Question 12

What is high-throughput sequencing

Answer 12

next generation sequencing

Question 13

What principle is used in microarray and in RNA sequencing

Answer 13

Hybridisation in microarray Direct sequencing in RNA

Question 14

Steps involved in microarrays

Answer 14

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

Question 15

What resolution is used in microarray and in RNA sequencing

Answer 15

several 100 bp in microarray single base in RNA

Question 16

What throughput is used in microarray and in RNA sequencing

Answer 16

high in microarray high RNA

Question 17

is the whole genome is used in microarray and in RNA sequencing

Answer 17

yes in microarray sometimes in RNA

Question 18

What level of background noise is there in microarray and in RNA sequencing

Answer 18

high in microarray low in RNA

Question 19

Are microarray and RNA sequencing capable of map transcription and DGE

Answer 19

Yes microarray Yes in RNA

Question 20

What is the dynamic range in microarray and RNA seq.

Answer 20

Up to a few hundredfold in microarray and >8000 fold

Question 21

Comapre distinguishng of isoform in microarray and RNA seq

Answer 21

Limited in microarray and yes in RNA seq

Question 22

Comapre distinguishng of alleic expression in microarray and RNA seq

Answer 22

Limited in microarray and yes in RNA seq

Question 23

Comapre amount of RNA amount required in microarray and RNA seq

Answer 23

high in microarray and low in RNA seq

Question 24

Compare cost for large genomesin microarray and RNA seq

Answer 24

high in microarray and relatively low in RNA seq

Question 25

Which company is dominant in terms of high throughput suquencing

Answer 25

Illumina

Question 26

What are the grounds for competitionin high throughput seuqncing

Answer 26

driven down cost, increased throughput

Question 27

Illumina Sequencing

Answer 27

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

Question 28

How do we sequence RNA

Answer 28

High througput sequencing technologies to get info about RNA content -> mRNA -> cDNA -> used for sequnecing library gen. -> allws quantification, profiling and discovery of RNA

Question 29

How does RNA-seq work?

Answer 29

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

Question 30

What are RNA-seq considerations

Answer 30

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

Question 31

What are other functional genomics approaches

Answer 31

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

Question 32

WHat is ChIP-Seq

Answer 32

Cross-link proteins to DNA?

Question 33

What occurs in ChIP-Seq

Answer 33

Isolate DNA and shear * Sonication for ‘random’ shearing * Immunoprecipitate protein of interest * Reverse cross-linking * Purify DNA * Sequence

Question 34

What is ATAC-Seq

Answer 34

Assay for Transposase-Accessible Chromatin

Question 35

What is ATAC-seq similar to

Answer 35

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

Question 36

What is bisulphite sequencing

Answer 36

Bisulphite treatment is used to determine the methylation state of DNA * Methylated cytosine protected from deamination * Unmethylated cytosine converted to thymine (via uracil)

Question 37

What occurs in bisulphite sequencing

Answer 37

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)

Question 38

Reduced Representation bisulphite sequencing (RRBS) - what causes this?

Answer 38

Human genome has 28m CpG sites and Assaying all of these in a single experiment problematic

Question 39

What enzyme does RRBS utilise to enrich for CpG?

Answer 39

Msp1

Question 40

What does Msp1 result in?

Answer 40

fragments which begin/end with CpG

Question 41

How much of the genome needs to be sequenced for RRBS?

Answer 41

1%

Question 42

High throughput sequencing produces ______ amounts of data

Answer 42

large

Question 43

Data can be ____ and complex

Answer 43

Noisy

Question 44

Computational approaches needed to make most of data for both

Answer 44

processing and analysis