Lecture 2

Question 1

what percentage of the DNA in the human genome actually codes for proteins

Answer 1

2%

Question 2

what is one of the most common genetic variations

Answer 2

at specific sites some people may have a G, while others may have an A

Question 3

what is NGS

Answer 3

next generation sequencing
it has the capacity to read DNA quickly and cheaply allowing for rapid identification of genetic variants

Question 4

sequencing a human genome using NGS

Answer 4

• genome is fragmented into small, random DNA pieces that are then sequenced separately.
• computer algorithm searches for overlapping sections within these DNA sequences
• algorithm assembles the fragments into their proper sequence,
• thus piecing the entire genome back together

Question 5

why has it become easier to sequence more genomes

Answer 5

most genomes share a high degree of similarity

Question 6

how are genetic variants identified

Answer 6

from an individual human genome sequence by comparing it to a reference

Question 7

next generation sequencing workflow

Answer 7

1. library preperation
2. cluster generation
3. sequencing
4. data analysis

Question 8

key steps in NGS of a human genome using sequencing by synthesis

Answer 8

• genome is fragmented into small, random DNA pieces
• each fragment is amplified to create clusters
• fragments are read by primers
  nucleotides are added to the DNA clusters
• camera captures fluorescent images emitted from each cluster
• the colour of the fluorescence identifies the nucleotide added

Question 9

flow cell

Answer 9

a thick glass slide with channels or lanes

clusters are generated on it

Question 10

why do we amplify the fragments

Answer 10

the fragments all represent part of the human genome and the goal is to read each od them individually

but it is very hard to read them singularly so we amplify them

Question 11

what is a cluster

Answer 11

bright spots on the image

• each cluster represents thousands of copies of the same DNA strand

Question 12

long reading sequencing

Answer 12

a DNA reading sequencing technique that enables thr sequencing of much longer DNA fragments than traditional short-read sequencing methods

• can provide better coverage of certain genomic regions
• is less accurate than short read sequencing

Question 13

locus heterogeneity

Answer 13

a genetic variant in one of several genes may lead to a clinically similar disease

Question 14

when sequencing a panel of genes associated with a clinical condition
which genes should be on your panel

Answer 14

genes with moderate to definitive disease association
genes with creative variants or additional data

Question 15

heterozygous variants

Answer 15

when a person has two different variants at a specific genetic location
( for example one from each parent)

Question 16

homozygous variants

Answer 16

when a person has two identical variants at a specific genetic location, either two normal variants or two diseased variants

Question 17

variant calling from sequencing data in a clinical context

Answer 17

1) the patients DNA is extracted and then sequenced
2) sequenced DNA fragments are then compared to a reference genome.
3) special software identifies any variants, which are differences between the sequences and reference genome
4) variants are interpreted to determine their potential significance