Next generation sequencing

Question 1

describe the human genome project

Answer 1

1990-2003
3 billion base pairs long
done with Sanger sequencing

Question 2

PCR

Answer 2

main principle for DNA sequencing application
PCR used to amplify specific region of DNA
each cycle doubles amount of DNA copies of your target sequence
amplify enough DNA molecules to have sufficient material to sequence or for other DNA applications

Question 3

Sanger sequencing

Answer 3

invented by Fred sanger
cycle sequencing
based on PCR
modified nucleotides

small proportion of free nucleotides are modified this way to allow every base in sequence to be read

one reaction = one sequence

Question 4

what is Sanger sequencing used for

Answer 4

identifying single nucleotide polymorphisms or mutations
identify monogenic disease-causing mutations
used for single gene tests

Question 5

next generation of DNA sequencing 1

Answer 5

technological advances since end of human genome projects

decrease In the cost of DNA

Question 6

NGS 2

Answer 6

development of new NSG methods began 13 years ago with 454 pyrosequencing

DNA sequencing throughput jumped 10 orders of magnitude

solexa sequencing by synthesis developed end of 2005

sequencing market to this day is now dominated by illumina SBS sequencing

Question 7

four steps of NGS sequencing

Answer 7

1. DNA library construction
2. cluster generation
3. sequencing by synthesis
4. data analysis

Question 8

step 1: DNA library construction

Answer 8

in wet lab we need to prepare DNA sample for sequencing
essentially DNA is chopped into small fragments = shearing
can be achieved chemically enzymatically or physically

Question 9

Step 1: DNA LIBRARY construction II

Answer 9

repair end of sheared DNA fragments
adenine nucleotide overhangs are added to end of fragments
adapters with thymine overhang can be ligated to DNA fragments
the end result is DNA library of billions of small stable random fragments, representative of our original DNA sample

Question 10

step 1: DNA library construction III

Answer 10

adapters contain essential components to allow library fragments to be sequenced
sequencing primer binding sites
p5 and P7 anchors for attachment of library fragments to the flow cell

Question 11

step 2: cluster generation

Answer 11

hybridise DNA library fragments to the flow cell
hybridisation to the flow cell is a random process
but we can’t measure individual single molecules of our DNA library-too small
we need to amplify the fragments to a bigger size that we can measure

Question 12

step 2: cluster generation 2

Answer 12

perform bridge amplification to generate clusters
many billions of clusters originating from single DNA library molecules
clusters are now big enough to be visualised
flow cell is now ready to be loaded on to the sequencing platform to perform the sequencing

Question 13

step 3: sequencing by synthesis I

Answer 13

modified 4 bases (ATCG) with chain terminators and diff fluorescent colour dye
sequence each single nucleotide

Question 14

step 3: sequencing by synthesis II

Answer 14

single nucleotide incorporation
flowcell wash
image the 4 bases
cleave terminator chemical group and dye with enzyme

Question 15

step 3: sequencing by synthesis III

Answer 15

camera sequentially images all 4 bases on the surface of the flow cell each cycle
each cycle image is converted to a nucleotide base call
cycle number anywhere between 50-600 nucleotide base pairs

Question 16

analysis of NGS data

Answer 16

short read sequences from the sequencing machine need to be re-assembled

mapping location of our sequence reads on the reference genome sequence

to generate consensus sequence of our original DNA sample library

Question 17

NGS v Sanger sequencing

Answer 17

NGS produces a digital readout

Sanger produces an analogue readout

NGS is a consensus sequence of many reads

Question 18

describe whole exome sequencing

Answer 18

21,000 genes in human genome

80% pathogenic mutations are protein coding

more efficient to only sequence the bits we are interested in, rather than the entire genome

Question 19

what does whole exome sequencing do

Answer 19

target enrichment
capture targets regions of interest with baits
potential to capture several Mb genomic regions of interest
exome would be 50Mb in size

Question 20

application of exome sequencing

Answer 20

collecting disease affected individuals and their families
use of NGS in disease gene identification
perform exome sequencing
compare variant profiles of affected individuals

try to identify the variant or mutation shared by the affected individual

Question 21

RNA-seq 1

Answer 21

NGS not just for studying DNA RNA-seq experiments use the total RNA from a collection of cells or tissue

RNA is first converted to cDNA prior library construction

NGS of RNA samples determine which genes are actively expressed

single experiment can capture the expression of thousands of genes

Question 22

RNA-seq II

Answer 22

number of sequencing reads produced from each gene can be used as a measure pf gene abundance

quantification of the expression levels

calculation of the difference in gene expression of all genes in the experimental conditions

with appropriate analysis, RNA-seq can be used to discover distinct isoforms of genes are deferentially regulated and expressed