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Genomics > Proteomics & Transcriptomics > Flashcards

Proteomics & Transcriptomics Flashcards

1
Q

Expression profiling

A

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

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2
Q

Microarrays

A

Assay expression of thousands of genes

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3
Q

Gene chips

A

slides or chips with oligonucleotides

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

multiple per gene;

attached to chip

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4
Q

How do microarrays show expression levels

A

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

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5
Q

Microarray – RNA-seq comparisons for expression profiling:

Microarray advantages

A

cheaper, less computational intensive than RNA-Seq

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6
Q

Microarray – RNA-seq comparisons for expression profiling:

Microarray Disadvantages

A
  • Need a reference genome or transcriptome to construct arrays
  • Need to verify results with qRT-PCR
  • Cross-hybridization to genes with highly similar sequences
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7
Q

Microarray – RNA-seq comparisons for expression profiling:

RNA-seq, advantages

A

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
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8
Q

Microarray – RNA-seq comparisons for expression profiling:

RNA-seq, disadvantages

A
  • Very expensive
  • Very large sequence files
  • Need scripting Knowledge (Python, R)
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9
Q

transcriptome profiling

A

Profiling: comparing expression levels in different samples

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11
Q

Transcriptome profiling platforms

A

Illumina is currently best

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12
Q

Transcriptome sequencing to get a reference transcriptome

A

Reference transcriptome sequencing:

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

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13
Q

RNA-seq data analysis

A

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

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14
Q

Measuring gene expression levels with Illumina RNA-seq

A

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

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15
Q

Ribosome profiling: Ribo-Seq

A
  • 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
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16
Q

Hi-C

A

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

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17
Q

How to study the three-dimensional architecture of genomes?

Steps of Hi-C?

A
  • 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
20
Q

Transcriptome sequencing platforms

A

454
Pacific Biosciences (PacBio)
Ion Torrent
Oxford

Nanopore’s Minion – longer reads

Short reads: Illumina

21
Q

Genomic studies of cis-regulatory elements

A

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

22
Q

DNase1-seq

A

Used to identify protein binding sites that are potential regulatory elements

23
Q

Chromatin immunoprecipitation followed by tiling array or Illumina sequencing

ChIP-chip or ChIP-seq

A

To analyzes binding sites of an individual TF

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

24
Q

DAP-seq

DNA affinity purification sequencing

A
  • 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
25
Q

How DNAse1-seq works

A
  • 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

26
Q

What DNase1-seq reveals

A
  • Reveals protein binding sites on a genome-wide scale at single base resolution
  • Can reveal new cis regulatory elements in genes
27
Q

How to ChIP-chip or ChIP-seq

A
  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!
28
Q

DAP-seq vs ChIP-seq

A

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
29
Q

How to DAP-Seq

A
  • 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
30
Q

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

A

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

31
Q

_______ identifies only ___ TF at a time

A

ChIP-seq identifies only 1 TF at a time

32
Q

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

A

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

33
Q

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

A

D) All are true

34
Q

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

A

B) It depends on genome annotation for mapping reads

35
Q

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

A

D) All of these

36
Q

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

A

D) All of these

37
Q

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

A

D) They can only identify previously known expressed genes

38
Q

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

A

C) Histone modifications can be identified

39
Q

What can be done with RNA-seq?

A

A) Alternatively spliced introns can be identified

B) Exon-intron boundaries can be identified

40
Q

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

A

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

41
Q

What are drawbacks of RNA-seq?

A

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

42
Q

liquid chromatography

A

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

Genomics (9 decks)

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