MBB 267 Week 3: RMC 5

Question 1

What is Genomics used for?

Answer 1

Used to identify the primary structure of the genome ( the sequence of As, Cs, Gs, and Ts, through de novo assembly or read mapping)

Question 2

What is transcriptomics used for?

Answer 2

Is the study of genome-wide gene expression, which is used to identify information about:

• What gene are expressed
• Time of expression
• the amount of expression

Question 3

What is a microarray?

Answer 3

A microarray is a laboratory tool used to detect the expression of thousands of genes at the same time.

Question 4

How does a microarray work?

Answer 4

Process
-it consists of a glass slide, onto which a spot consisting of lots of copies of a probe sequence can be attached, the copies of the probes are synthesised on the surface of the array.
-The microarray consists of many spots in an ordered arrangement , and the exact position of each spot is known. Each spot has a different sequence, which has been designed to correspond to a particular region of a genome of interest.
-We hybridise a fluorescently labelled RNA sample to a probe on the surface of the microarray.
-If a particular transcript has a complementary sequence to one of the probes on the array, it will hybridise to it. —–The fluorescence can be detected at that particular position on the surface of the array.
The amount of transcript which hybridises to a particular spot is proportional to the abundance of that transcript in the RNA sample - so spots with sequences which correspond to highly expressed genes will show bright fluorescence, while weak expressed genes will show less fluorescence, and genes which are switched off will show little or no sign

Question 5

What are the limitations of Microarrays?

Answer 5

Considered as a low-resolution sequencing technology

• If we get a signal for a particular probe, we know that that sequence (or something sufficiently similar) is present in our sample. However, we usually don’t know if that is the exact sequence that is present
• Only known sequences are covered by the microarray, unknown sequences are not covered
• limit to how much RNA can hybridise to a particular spot on the microarray, can limit highly expressed genes

Question 6

What is an RNA-seq?

Answer 6

RNA-seq is a technique that can examine the quantity and sequences of RNA in a sample using next generation sequencing. It analyzes the transcriptome of gene expression patterns encoded within RNA.

Question 7

How does RNA-seq work?

Answer 7

RNA extracts from our cells can be fragmented and converted to cDNA. We can then attach adapters to the ends of our fragments, and sequence them on an Illumina, to produce RNA-seq reads. The RNA-seq reads produced by the Illumina can be mapped to a reference genome. The number of reads mapping to each gene is used as a measure of the expression level of each gene

Question 8

What is the use of Microarrays and RNA-seq?

Answer 8

Microarrays and RNA-seq allow us to perform genome-wide experiments to measure the expression of every gene simultaneously

Question 9

What is a Northern Blot?

Answer 9

A traditional method of assessing expression of a particular gene. Radiolabelled probes are used to detect the presence of a particular transcript within a whole cell RNA extract. The level of expression can be assessed quantitatively by the size of the band

Question 10

What is RT-qPCR?

Answer 10

Reverse transcription quantitative PCR( RT-PCR) uses reverse transcriptase to make cDNA from transcripts.

Question 11

How does RT-qPCR work?

Answer 11

Process of how RT-qPCR works?

• We have our RNA sequence and we reverse transcribe it, forming cDNA.
• The cDNA corresponding can be PCR amplified,
• Then use fluorescent primers, which allows the level of the transcript to be quantified relative to a reference gene or a DNA standard.

Question 12

What is the challenge faced when using RNA-seq on eukaryotic organisms?

Answer 12

The processed mRNAs consist of adjacent exon sequences, but the exons are separated by introns in the genome sequence. Reads overlapping exon junctions need to be split during mapping. To do this we need a splice aware aligner.

Question 13

What is De novo assembly?

Answer 13

De novo sequencing refers to sequencing a novel genome where there is no reference sequence available for alignment. This is similar to genome assembly, but more complex since not all transcripts are present at the same level, and the same gene may produce multiple different transcripts

Question 14

RNA-seq vs. Microarrays

Answer 14

• Similarities:
• both well-developed technologies with minimal technical variation
• Differences:
• RNA-seq has a larger dynamic range than microarrays i.e. a greater ability to distinguish different levels of expression
• Microarrays only give information for pre-selected regions of the genome. RNA-seq is genome-wide, and can detect novel transcripts
• RNA-seq allows us to detect differences from the reference genome, such as SNPs in transcribed regions
• RNA-seq can be done without a reference genome – de novo assembly of the transcriptome is possible

Question 15

What are Technical Replicates?

Answer 15

involve assessing the same biological sample multiple times. Tells us about technical variation in our microarray or RNA-seq procedure

Question 16

What are Biological Replicates?

Answer 16

requires us to repeat the entire experiment independently. Tells us about the biological variation in the system. Typically biological variation is larger than technical variation, so it is usually appropriate to perform multiple biological replicates.

Question 17

What is Chromatin Immunoprecipitation (ChIP)?

Answer 17

ChIP is a method that can be used to isolate DNA bound by a specific protein

Question 18

How does ChIP work?

Answer 18

So;

• Proteins covalently crosslinked to DNA by treating with formaldehyde.
• Chromatin sheared by sonication or using an endonuclease. Use of an exonuclease (ChIP-exo) allows the bound DNA to be trimmed to the binding site.
• Immunoprecipitation and purification of bound DNA using an antibody specific to the protein of interest

Question 19

How does ChIP-seq work?

Answer 19

A 5’3’ exonuclease is used to trim the DNA before the binding site. This means that there is an offset between reads on the forward and reverse strand, which allows the exact boundaries of the binding site to be determined. Reads are mapped to the reference genome, and binding sites are identified as peaks in the signal.

Question 20

What is Chromosome Conformation Capture?

Answer 20

Chromosome conformation capture techniques are methods used to analyze the spatial organization of chromatin in a cell (to study chromatin structure)

Question 21

How does Chromosome Conformation Capture work?

Answer 21

Chromosome Conformation Capture methods use formaldehyde to form cross-links, cross-links are formed between interacting regions of the genome. We can digest the cross-linked chromatin, ligate the loose ends and then remove the cross-link to form a single continuous piece of DNA containing sequence from the two interacting regions.

Question 22

What are the different Chromosome Conformation Capture methods?

Answer 22

The methods are 3C, 4C, 5C and Hi-C. They differ in how they characterise DNA fragments

Question 23

What is Chromosome Conformation Capture 3C used for?

Answer 23

In 3C we look for specific interactions between two known partners. 3C uses two specific primers, so is good for targeting interactions between two known loci

Question 24

What is Chromosome Conformation Capture 4C used for?

Answer 24

4C allows us to identify remote regions which interact with one region of interest. 4C introduces a circularisation step, meaning that only one of the interaction partners needs to be pre-selected

Question 25

What is Chromosome Conformation Capture 5C used for?

Answer 25

5C allows discovery of novel interactions. 5C uses amplification using universal primers to allow interactions between uncharacterised loci to be identified.

Question 26

What is Chromosome Conformation Capture Hi-C used for?

Answer 26

allows comprehensive genome-wide characterisation of all of the interactions between remote chromosomal regions. Hi-C uses biotin-enrichment followed by high-throughput sequencing to get a comprehensive genome-wide view of all long range chromosomal interactions