microarrays

Question 1

is BCS-WGS still used in methylations studies if the Illumina infinitum BeadChip array outperforms it?

Answer 1

methylation microarrays such as the infinium bead chip array queries only a small proportion of CpG sites in the genome, although its at high precision, its limited to known/common CpG sites (content) by using probes whereas BCS-WGS can identify novel CpG sites and query way more CpG sites across the genome.

Question 2

are microarrays high or low throughput

Answer 2

high throughput

Question 3

what is the affymetrix gene chip

Answer 3

a chip the size of a thumbnail with 6.5 million sections with millions of identical probes
- 25bps in length

Question 4

what are the steps involved in using the affymetrix gene chip

Answer 4

RNA –> cDNA –> cRNA
cDNA generated for amplification (ds)
cDNA generated with biotinylated uracil NTs
cRNA is then formed by degrading the first strand leaving only the biotintylated strands as a probe

Question 5

what is the purpose of introducing biotinylated NTs into the affymetrix cDNA assembly

Answer 5

biotintylation gives marks on each fragment boosting the sensitivity making smaller frags/ lowly expressed genes or less sensitive fluorescent readouts to be read easier

Question 6

what is the normalisation of data

Answer 6

the minimisation of variation between samples between batches or even experiments allowing for easier analysis and comparison

Question 7

give examples of why must data be normalised

Answer 7

• different starting quantities of sample
• difference in labelling efficiencies in dyes used
• unequal probe hybridisation
• batch differences

Question 8

name some advantages of RNAseq vs Microarray

Answer 8

• RNAseq has ability to detect new transcripts, not limited by content
• can quantify expression with 100x wider dynamic range
• arrays are limited. to background at the low end and signal saturation at the high end
• higher spec. and Sens. as it can detect a higher percentage of diff. expressed genes, especially lowly expressed ones.

Question 9

what are the potential technical confounders for microarrays

Answer 9

• DNA quality (QC)
• Bisulphite conversion efficiency (QC)
• Batch effects (Randomise Samples)
• Position on plate (automation/randomise)
• Probe bias (normalisation)

Question 10

what are the potential biological confounders fo rmicroarrays

Answer 10

• genetics (muations, snps, cnvs)
• gender (prevalence, x-linked)
• cell composition (select correct cell for questioning)
• environmental exposures (e.g. control/exclude smokers etc.)
• therapies (drug interactions)

Question 11

what is a beta matrix

Answer 11

its a grid consisting of all CpG sites analysed as well as their corresponding methylation value