Proteomics & Transcriptomics

Question 1

Expression profiling

Answer 1

Comparing gene expression levels in 2 or more samples using thousands of genes

Question 2

Microarrays

Answer 2

Assay expression of thousands of genes

Question 3

Gene chips

Answer 3

slides or chips with oligonucleotides

either ~25 bp, ~70bp, correspond to each gene;

multiple per gene;

attached to chip

Question 4

How do microarrays show expression levels

Answer 4

cDNAs from 2 samples are fluorescently labelled with different dyes & competitively hybridized to gene chips

cDNA hybridized to chips, chips are scanned & fluorescence detected; data analyzed to determine expression level

Question 5

Microarray – RNA-seq comparisons for expression profiling:

Microarray advantages

Answer 5

cheaper, less computational intensive than RNA-Seq

Question 6

Microarray – RNA-seq comparisons for expression profiling:

Microarray Disadvantages

Answer 6

• Need a reference genome or transcriptome to construct arrays
• Need to verify results with qRT-PCR
• Cross-hybridization to genes with highly similar sequences

Question 7

Microarray – RNA-seq comparisons for expression profiling:

RNA-seq, advantages

Answer 7

can detect…

• rare transcripts, can detect
• allele-specific expression
• exon-intron boundaries
• alternative splicing

Don’t need reference Genome or transcript one
Don’t need to construct an array

• Works with non-model organisms
• No cross hybridization but can Multi-map reads

Question 8

Microarray – RNA-seq comparisons for expression profiling:

RNA-seq, disadvantages

Answer 8

• Very expensive
• Very large sequence files
• Need scripting Knowledge (Python, R)

Question 9

transcriptome profiling

Answer 9

Profiling: comparing expression levels in different samples

Question 11

Transcriptome profiling platforms

Answer 11

Illumina is currently best

Question 12

Transcriptome sequencing to get a reference transcriptome

Answer 12

Reference transcriptome sequencing:

all the RNAs present in the sample, multiple tissue/organs are used – can mix or not mix

Question 13

RNA-seq data analysis

Answer 13

1) Align reads to the genome or reference transcriptome
2) count reads
3) quantify expression for profiling using analysis programs

For read alignment: Bowtie (older), GSNAP, STAR
Cufflinks (older) — used to estimate transcript abundance & test for differential expression

Question 14

Measuring gene expression levels with Illumina RNA-seq

Answer 14

RNA-seq reads aligned to a genome: read coverage in exons is often not uniform

Normalize the number of reads to the length of the gene

Question 15

Ribosome profiling: Ribo-Seq

Answer 15

• Capture ribosome-mRNA transcript complexes in action
  translating mRNAs, by cycloheximide treatment
• 28-30 nt of a transcript captured
• Allows for high-throughput analysis of translational activity
• Measure the rate of protein production, but not abundance in contrast to MS proteomics

Question 16

Hi-C

Answer 16

Method to study the three-dimensional architecture of genomes by sequencing long range interactions of DNA regions

Question 17

How to study the three-dimensional architecture of genomes?

Steps of Hi-C?

Answer 17

• Cells are fixed with formaldehyde, causes interacting loci to be bound to one another by covalent DNA-protein cross links
• DNA is digested with a restriction enzyme, the linked loci remain intact
• Blunt-end ligation is performed to ligate cross linked DNA fragments
• Results in genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus
• Library is sheared, junctions are pulled down with streptavidin beads (bind to biotin)
• Purified junctions are sequenced

Question 20

Transcriptome sequencing platforms

Answer 20

454
Pacific Biosciences (PacBio)
Ion Torrent
Oxford

Nanopore’s Minion – longer reads

Short reads: Illumina

Question 21

Genomic studies of cis-regulatory elements

Answer 21

DNase1-seq: identifies protein binding sites that are potential regulatory elements

Chromatin immunoprecipitation followed by tiling array (ChIP-chip) or Illumina sequencing (ChIP-seq): analyzes binding sites of an individual TF

DAP-seq: DNA affinity purification sequencing

Question 22

DNase1-seq

Answer 22

Used to identify protein binding sites that are potential regulatory elements

Question 23

Chromatin immunoprecipitation followed by tiling array or Illumina sequencing

ChIP-chip or ChIP-seq

Answer 23

To analyzes binding sites of an individual TF

To characterize histone modifications
- Can reveal his tone marks in gene coding vs. regulatory sequences

Question 24

DAP-seq

DNA affinity purification sequencing

Answer 24

• TF binding site discovery assay
• Couples affinity-purified TFs with Illumina sequencing of
  a gDNA library
• Identifies genome-wide binding locations for each TF assayed

Question 25

How DNAse1-seq works

Answer 25

• DNase 1 cuts at open chromatin sites where no protein is bound, locations of bound proteins are protected
• Sequencing DNA to show sites of bound proteins

Assumes bound proteins are regulatory factors
- Does not reveal identity of the proteins

Question 26

What DNase1-seq reveals

Answer 26

• Reveals protein binding sites on a genome-wide scale at single base resolution
• Can reveal new cis regulatory elements in genes

Question 27

How to ChIP-chip or ChIP-seq

Answer 27

1. Cross-link complex with formaldehyde
2. Purify nuclei
3. Fragment chromatin proteins bound to DNA
4. Immuno-enrich protein-DNA complex antibody to a specific TF
5. Reverse cross-links (65 degrees C)
6. Hybridized to array or Illumina Sequence!

Question 28

DAP-seq vs ChIP-seq

Answer 28

DAP-Seq: - Faster, less expensive, more easily scaled up than ChIP-seq - DNA library constructed using native gDNA from any source, preserves cell and tissue specific chemical modifications that can affect TF binding such as DNA methylation

Question 29

How to DAP-Seq

Answer 29

- DNA library is incubated with affinity-tagged in vitro expressed TF - TF-DNA complexes are purified using magnetic separation of the affinity tag - Bound genomic DNA is eluted from TF and sequenced - Identifies genome-wide binding locations for each TF assayed

Question 30

__________ and ________ ID TFs that bind regulatory elements, but _________ does not

Answer 30

ChIP-seq and DAP-seq ID TFs that bind regulatory elements, but DNase1 does not

Question 31

_______ identifies only ___ TF at a time

Answer 31

ChIP-seq identifies only 1 TF at a time

Question 32

DNase1-seq identifies all __________________ in a genome but not _________________.

Answer 32

DNase1-seq identifies all TF binding sites in a genome but not TFs that bind them

Question 33

Which of the following is not true about microarrays? A) Transcripts hybridize with the probes on the array B) Multiple oligonucleotide probes are designed for each gene C) Transcripts are labeled with fluorescent dyes D) All are true

Answer 33

D) All are true

Question 34

Which of the following is not true about RNA deep sequencing (RNA-seq) A) It can detect currently unrecognized expressed genes B) It depends on genome annotation for mapping reads C) It can detect allele-specific expression D) All are true

Answer 34

B) It depends on genome annotation for mapping reads

Question 35

Which of the following are drawbacks of RNA-seq? A) Large sequence file sizes B) High cost C) Uneven sequencing depth along the length of a transcript D) All of these

Answer 35

D) All of these

Question 36

Which of the following are disadvantages of expressed sequence tag (EST) sequencing? A) It generally cannot identify mRNAs that are expressed at low levels B) It is relatively slow C) It is expensive for the amount of data acquired D) All of these

Answer 36

D) All of these

Question 37

Which of the following is not a drawback of tiling microarrays (which are different from regular microarrays)? A) They may not distinguish between highly similar duplicated genes B) They may not identify lowly expressed genes C) The organism must have a sequenced genome D) They can only identify previously known expressed genes

Answer 37

D) They can only identify previously known expressed genes

Question 38

Which of the following can not be done with RNA-seq? A) Alternatively spliced introns can be identified B) Exon-intron boundaries can be identified C) Histone modifications can be identified D) All of these can be done

Answer 38

C) Histone modifications can be identified

Question 39

What can be done with RNA-seq?

Answer 39

A) Alternatively spliced introns can be identified | B) Exon-intron boundaries can be identified

Question 40

What are disadvantages of expressed sequence tag (EST) sequencing?

Answer 40

A) EST sequencing generally cannot identify mRNAs that are expressed at low levels B) It is relatively slow C) It is expensive for the amount of data acquired

Question 41

What are drawbacks of RNA-seq?

Answer 41

A) Large sequence file sizes B) High cost C) Uneven sequencing depth along the length of a transcript

Question 42

liquid chromatography

Answer 42

Separate proteins by 2D gels, or liquid chromatography, cut out spots, elute proteins, digest into peptides with trypsin protease