Bioinformatics

Question 1

How Illumina sequencing works

Answer 1

1. DNA sample is fragmented
2. DNA hybridizes with slide-bound adapters
3. Fluorescently-labeled bases are added to growing DNA based off of primers
4. When base is added to new DNA strand, camera captures fluorescence given off
5. Fluorophore is cleaved and 3’-OH group is regenerated
6. Reaction continues until DNA is completely synthesized

Question 2

Results of Illumina sequencing

Answer 2

Hundreds of millions of short reads

Results can be single-end or paired-end (DNA is read twice, from opposite sides)

Question 3

Limitations of Illumina sequencing

Answer 3

Errors can occur (PCR errors, etc.)
Long insertions and deletions can be difficult to call from short reads
Files produced are very large

Question 4

Exome sequencing

Answer 4

Sequencing exons rather than all DNA: variation in exons is easier to interpret than in non-coding regions

Question 5

Variants searched for in exome sequencing: characteristics a disease variant would have

Answer 5

Present in proband, but not parents (mutations occurred)
Uncommon variants (when comparing proband with individuals lacking disease, common variants probably wouldn't cause rare disease)

Question 6

How to tell whether or not a gene is crucial/disease causing

Answer 6

Genes with few mutations are more likely to be crucial (person wouldn’t survive without functional gene)
When gene is mutated, it is more likely to cause disease