CMB2000/L16 Functional Genomics Flashcards
Give 3 different types of genomics experiments.
Protein/DNA interactions
DNA methylation
Gene expression
Protein-protein interactions
Loss-of-function
Describe microarrays. (3)
Measurement of hybridisation
Sample to probes on array
Range of samples and probes for different experiment types
Explain how microarrays work. (4)
Sample preparation - RNA extraction converted to cDNA
Labelled with fluorescent dyes
Hybridisation of cDNA to probes on microarray
Scanning of fluorescence proportional to cDNA on probes
What can be used as a substitute for hybridisation? Give an advantage.
Direct sequencing
Gives greater resolution and accuracy
Give a disadvantage to using direct sequencing in the place of hybridisation.
Cost and throughput
Give another name for high-throughput sequencing.
Next generation sequencing
Describe how Illumina sequencing works. (5)
Fragments of DNA bound to solid surface
Solid-phase bridge PCR forms clonal clusters
Sequencing proceeds in cycles
Modified nucleotides with fluorescent group blocks extension
Reversible termination allows sequencing to proceed to next cycle
How does the fluorescent group of modified nucleotides affect adding of bases?
Only 1 base can be added per cycle
Different fluorophore per base
Describe step 1: sample preparation of Illumina sequencing. (2)
DNA fragmentation
Adapter ligation - short known sequences added to ends of DNA fragments- known for binding to flow cell and amplification/sequencing
Describe step 2: cluster generation of Illumina sequencing. (2)
Flow cell loading - prepared DNA library loaded onto flow cell (glass slide with channels coated in complementary oligonucleotides to adapters)
Bridge amplification - each fragment binds to oligonucleotides and fragment being amplified
Describe step 3: sequencing by synthesis of Illumina sequencing. (4)
Incorporation of nucleotides with fluorescence
Imaging with high-res camera
Cleavage of fluorescent label and terminating group
Repetition for 100-300 cycles
Describe step 4: data analysis of Illumina sequencing. (3)
Base calling - processing of images to determine sequence of fragments
Alignment and assembly - alignment to reference genome and assembled de novo
Variant calling - identification of variants e.g., SNPs, indels
Describe RNA sequencing. (4)
High-throughput sequencing technologies to get information about RNA content
RNA converted to cDNA
cDNA used to sequencing library generation
Allows quantification, profiling and discovery of RNA
Compare RNA-Seq to microarrays.
RNA-Seq
Based on direct sequencing over hybridisation
Single base resolution
High throughput
Sometimes need genome
Lower background noise
Microarrays
Hybridisation
Several - 100bp resolution
Need genome
High background noise
Describe the process of RNA-Seq. (6)
Poly-A selection
Fragmentation
Random priming
First and second strand cDNA synthesis
End repair, phosphorylation and A-tailing
Adapter ligation, PCR amplification and sequencing
Give 3 considerations of RNA-Seq.
Big data sets need expert processing
Expression data can be noisy
Easy for confounding factors to dominate
Good practise same as for any statistical approach
What is the general idea of many functional genomics approaches?
Aspects of nucleic acid biochemistry based on sequencing
Enrich specific molecules or regions of molecules and sequence
Analyse to show what’s been enriched
Describe the process of ChIP-Seq. (6)
Cross-link proteins to DNA (in-cell)
Isolate DNA and shear (sonication for random sharing)
Immunoprecipitate protein of interest
Reverse cross-linking
Purify DNA
Sequence
What does ATAC-Seq stand for?
Assay for Transposase-Accessible Chromatin
Describe ATAC-Seq.
Similar to DNAse-Seq
Relies of transposase Tn5
Adapter ligated fragments isolated, amplified and sequenced
Describe transposase Tn5 and give an example of where it is used.
High activity
Highly efficient cutting of exposed DNA
Ligation of adapters to ends
Used in ATAC-Seq
Describe bisulphite sequencing (BS-Seq) (3).
Used to determine methylation state of DNA
Methylated cytosine protected from deamination
Unmethylated cytosine converted to thymine (via uracil)
What are hyper-methylated DNA regions associated with?
Transcriptional silencing
Describe Reduced Representation BS-Seq. (3)
Uses Mspl restriction enzyme to enrich for CpGs
Recognition site C/CGG
Results in fragments which begin/end with CpG
Only need to sequence 1% of genome
