Next generation sequencing

Question 1

Explain the human genome project

Answer 1

Human Genome Project (1990 -
2003)
* 3 billion base pairs long
* All done with traditional Sanger
Sequencing
* Unravelled the first Human Genome
Sequence to drive genetics research
* Billion-dollar cost

Question 2

Explain the principles of PCR

Answer 2

Fundamental principle for any DNA
sequencing application
3 step process
* PCR is used to amplify a specific
region of DNA; primers flank the
region you want to amplify.
* Each cycle doubles the amount of
DNA copies of your target sequence
https://www.shmoop.com/dna/pcr-polymerase-chain-reaction.html
* Amplify enough DNA molecules

Question 3

Explain characteristics of Sanger sequencing

Answer 3

Invented by Fred Sanger in 1977
* Cycle Sequencing
* Based on PCR (and needs a PCR product
as input)
* Identify single nucleotide
polymorphisms (SNPs), or
mutations
* We can identify monogenic
disease-causing mutations
* Used often for single gene tests
* E.g. CTFR in cystic fibrosis Needs Primers
* Modified nucleotides
● Chain Terminators
● Nucleotide specific colour tag
* A small proportion of the free nucleotides
are modified this way to allow every base
in the sequence to be read

Question 4

Explain the next generation of DNA sequencing I

Answer 4

-technological advances since the end of the human genome project
-decrease in the cost of DNA sequences
-the cost has dropped at a rate faster than that of Moore’s law since the end of 2007

Question 5

Explain the next generation of DNA sequencing II

Answer 5

-development of new NGS methods began 13 years ago with 454 pyrosequencing
-DNA sequencing throughout jumped 10 orders of magnitude
-solexa sequencing by synthesis (SBS) developed end of 2005

Question 6

Outline the 4 steps in NGS sequencing

Answer 6

1) DNA library construction
2) Cluster generation
3) Sequencing by synthesis
4) Data analysis

Question 7

Explain DNA library construction

Answer 7

1) DNA library construction:
- in the wet lab- we need to prepare DNA sample for sequencing
-DNA is chopped into small fragments—> this is called shearing
-this can be achieved chemically, enzymatically or physically

2) We have to repair the end of shared DNA fragments
-Adenine nucleotide overhangs are added to the end of fragments
-Adapters with thymine overhangs can be ligated to the DNA fragments

3) Adapters contain the essential components to allow the library fragments to be sequenced
-sequencing primer binding sites
-P5 and P7 anchors for attachment of library fragments to the flow cell

Question 8

Explain cluster generation

Answer 8

Cluster generation:
-hybridise DNA library fragments to the flow cell
-we need to amplify the fragments to a bigger size for a stronger signal

-perform bride amplification to generate clusters
-many billions of clusters originating from single DNA library molecules

-clusters are now big enough to be visualised and the flow cell is now ready to be loaded

Question 9

Explain sequencing the library

Answer 9

-DNA libraries deposited on flow cell:
- bridge amplification
-amplified to form ‘clusters’

Question 10

Explain sequencing by synthesis

Answer 10

Sequencing by synthesis:
-modified 4 bases (ATCG) with:
* chain terminators
* different fluorescent colour dye

-sequence each single nucleotide 1 cycle at a time in a controlled manner

-single nucleotide incorporation (DNA polymerase)
-flow cell wash
-image the 4 bases
-cleave terminator chemical group and dye with enzyme \

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

-there are millions of short read sequences representing our original DNA library

Question 11

Explain analysis of NGS data

Answer 11

-short read sequences from the sequencing machine need to be pieced together like a jigsaw
-mapping locations of our sequence reads on the reference genome sequence
-to generate a consensus sequence of our original DNA sample library

Question 12

Explain NGS vs Sanger

Answer 12

NGS produces a digital readout. Sanger produces an analogue readout

Sanger is one sequence read

NGS is a consensus sequence of many reads

Question 13

Explain whole genome sequencing

Answer 13

-there are roughly 21,000 genes in the human genome
-often we are only interested in the gene protein coding exons or the ‘exome’ represents 1-2% of the genome

-some 80% pathogenic mutations are protein coding

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

-patient DNA sample subjected to exome sequencing

Question 14

Explain application of exome sequencing

Answer 14

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

Question 15

Explain RNA sequencing

Answer 15

-RNA- seq experiments use the total RNA (or mRNA) from a collection of cells or tissue

-RNA is first converted to cDNA prior to library construction
-NGS or RNA samples determine which genes are actively expressed

-the number of sequencing reads produced from each gene can be used as a measure of gene abundance
-quantification of the expression levels
-calculation of the differences in gene expression of all genes in the experimental conditions.