Omics Flashcards
What are the 5 omes of the genome?
Which techniques can be used for each?
Epigenome, trancriptome, proteome, metabolome, microbiome
Epigenome = DNA seq, liquid chromatography-MS or MS Transcriptome = DNA seq Proteome = LC-MS/MS Metabolome = LC-MS/MS Microbiome = DNA seq
What is the transcriptome?
What is a gene signature?
Which RNAs are involved in protein synthesis?
Which RNAs are involved in post-transcriptional modification/DNA replication?
All the transcripts in a genome (so all the expressed genes)
Group genes in a cell with uniquely characteristic pattern of gene expression that can result in altered or unaltered biological process/medical condition
Coding RNA so mRNA & pre-mRNA
snRNA & snoRNA
3 methods of extracting RNA are: phenol chloroform, column based & bead based. What are the steps and their advantages/disadvantages?
How can you quantify RNA? (2 ways)
How can you measure RNA quality? What’s the score system?
- = guanidinium thiocyanate & phase separation so RNA precipitated out.
Quick, low cost & high yield. Low purity - High purity. Low yield & high cost
- Lyse tissue with guanidine, add to a column & spin so RNA & DNA stick to the filter. Wash protein away & add DNA to remove DNA. Add water to loot off RNA. High cost. Highly pure & automated.
- UV spectroscopy 260/280 ratio with quartz cuvette- Nanodrop/Denovix & Beer-Lambert
- Fluorogenic dyes bind selectively to RNA
28S/18S ratio: run an agarose gel & measure with tape station. 1-10 where 6+ is good quality
How can you analyse gene expression with RNA?
Why is this method less favourable?
How can this be improved?
What is cDNA mixed with and how is it visualised in qPCR?
How is the data normalised?
What technique has replaced RNA sequencing?
Northern Blotting
RNA is less stable than cDNA & it’s not quantitative
Reverse transcribe the mRNA into cDNA and then PCR for specific target
Target specific primers & polymerase Taq
- Probes or Sybr green
With a housekeeping gene
Microarrays
How could you prepare a DNA library easily?
Why are these limited?
What is 2nd gen sequencing?
3rd gen?
With a kit
storage time of cDNA, quantity of RNA & cyropreservation of cell samples
Short reads (36-200bp)- sequencing with ligation or synthesis
Long reads (up 20kbp)- Real time sequencing & synthetic approaches
Next generation sequencing:
Illumina Genome Analyser Sequencing by synthesis- how does it work?
Single cell sequencing- how does it work?
What are essentially the 2 advances of next generation sequencing and what are their advantages?
Templates hybridise to ‘flow cell slide bound adapters’ & interact with nearby primers. Nucleotides added in amplification and imagine-colour determines what base is added in a cluster.
Measuring transcriptome in bulk tissue. Cells in droplets go through flow cell to prepare libraries.
Short read- low cost, high accuracy, good for large studies
Long read- read lengths good for de novo
What is a reference genome?
What is it useful for?
How is FASTA different to FastQ?
How is SAM different to FASTA?
How is BAM different to SAM?
Collection of contigs (overlapping DNA reads) in FASTA
Genes to be evaluated in genomic context- used to align high throughput sequencing in a map of an organism
Unaligned sequences (same) but with quality
Aligned sequences
Compressed SAM file
What are the statistical challenges with RNA sequencing analysis?
How could you do analysis?
Very high dimensional data, small sample size, can’t do a T test as includes 30k genes in a parameter
In R
What is proteomics?
What are the traditional techniques?
What are the techniques now? Why is this useful?
Large scale study of proteins
SDS-page & Western blotting
Liquid chromatography mass spec- measure many proteins at once
How are the 3 ways to prepare proteins for mass spec?
What is the critical step after to attain peptides?
Why is trypsin used to digest proteins?
What happens after to the tryptic peptides?
Peptides of different m/z are seen in MS1, what happens to the most dominant peptides?
What do the peaks in the 2nd spectra show?
In gel digestion- de-stain gel & dehydrate in acetonitrile & incubate in trypsin
In solution digestion- lysis (reducing agents), solvent precipitation (acetone/methanol), re-suspend in urea & incubate with trypsin
Filter aided digestion- Lysis & then wash in urea & ammonium bicarbonate
De-salt with reverse phase chromatography
Makes short peptides (as cleaves C terminal lysine & arginine)
Acidified (for a positive charge) & injected into HPLC column & sent to mass spectrometer
Fragment with inert gases to cleave the peptide bonds (to form 2 fragments with collision induce peptide bonds) to form MS2.
Peaks of N-terminal fragments superimposed over peaks of C-terminal fragments
What is a proteomics interactome used for?
What are the steps of making one?
How can you ensure that the protein interaction is real (not just with denatured proteins)?
What post-translational modification could you make to purify specific proteins?
What proportion of kinases found make up the phosphoproteome?
Identifying protein-protein interactions
- Use antibody/expression tag to bind to protein of interest
- Also bind proximity labels such as BioID (use biotin ligase to label interacting proteins)
- Elute, prep the sample with trypsin, desalt & run LC-MS
Incubate for longer
Phosphorylation- IMAC bead with ligand attached will interact with phosphates on a peptide.
Then incubate, wash & elute with ammonia- end up with purifying only phosphopeptides
5%- and 150 don’t have known substrate
What is metabolomics?
What are the 2 main techniques?
What is the difference between analysing untargeted and targeted metabolomics?
What is the benefit to network analysis?
What are the + and - of omics data?
Study/analysis of metabolite composition (lipids, fatty acids, bile acids, polar metabolites etc) of cell type/tissue/biological fluid
NMR (less sensitive) & LC-MS (high sensitivity)
Un = analysis of all measurable analytes in sample. Tar = measurement of define groups of characterised & annotated metabolites
Overlap lots of omics data in multi-omics
\+ = snapshot global changes in proteins, post modifications, transcripts & metabolites - = sample prep & equipment can bias sample, narrow view, expensive
