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
