Next Generation Sequencing

Question 1

Recap: PCR

Answer 1

• Fundamental for any DNA seq application
• Three states - Denaturation, Annealing and Native state
• PCR is used to amplify a specific region of DNA - primers will flank the region you want to ampifly
• Each cycles doubles amount of DNA copies in target seq
• Amplify enough DNA molecules so that we have material for other applications

Question 2

How has NGS progressed with the advent of technology?

Answer 2

Technological advances since the end of the human genome project have decreased the cost of DNA sequencing

NGS has replaced sanger sequencing for almost all sequencing in the lab including Whole genome sequencing + Whole exome sequencing

Question 3

What are the four sequencing core principles?

Answer 3

1) DNA library construction
2) Cluster Generation
3) Sequencing-by-synthesis
4) Data Analysis

Question 4

What is a DNA library?

Answer 4

• A DNA library is a collection of random DNA fragments used for further study (in this case NGS)
• These are generally derived from patients blood

Question 5

Describe the first step of NGS

Answer 5

DNA library construction

1. Wet lab prepares DNA sample for sequencing
2. DNA is chopped into small fragments via shearing.This can be achieved by chemically, enzymatically or sonication
3. Sheared ends are repaired by adding adenine (A) nucleotide overhangs to the end of the fragments via a process called A-tailing
4. This allows adapters with thymine (T) to be ligated to the DNA fragments
5. The result is a DNA library with small, stable random fragments representative of the original DNA sample
6. Adapters contain all the essential components to allow the library fragments to be sequenced such as sequencing primer binding sites
7. Adaptors contain P5 and P7 anchors which allow for attachment of library fragments to the flow cell

Question 6

Describe the second step of NGS

Answer 6

Cluster Generation

1. DNA library fragments are hybridised to the flow cell (this is a random process)
2. The single molecules of the DNA library are too small therefore they are amplified by bridge amplification PCR to generate CLUSTERS
3. This allows clusters to be large enough to visuallise and flow cells are ready to be loaded onto a sequencing platform

Question 7

What is the third step of NGS?

Answer 7

Sequencing-by-synthesis

1. One nucleotide is sequenced per 1 cycle in a controlled manner
2. This is carried out using 4 modified bases (ATGC) with chain terminators and different fluorescent colour dye
   
   1. Incorporate nucleotide with polymerase
   2. Flow cell wash
   3. Image the base
   4. Cleave the terminator chemical group and dye with enzyme
   5. Repeat (n) times for full seq. length

• A camera will sequentially image all 4 bases on the surface of the flow cell in each cycle
• Each cycle image is converted into a nucleotide base call (AGGT)
• Short read sequences will represent the original DNA library

Question 8

Explain the fourth step of NGS

Answer 8

Data Analysis

1. Short read sequences are reassembled like a jigsaw to generate a consensus sequence of the original sample
2. The short read sequences align and map against the reference sequence
3. The consensus sequence is compared against the human genome reference and looks for genetic variants
4. Dedicated software and bioinformatic tools will achieve this

Question 9

What is the most common application of NGS?

Answer 9

Exome Sequencing

• 21,000 genes in the human genome, however only 2% of the genome codes for protein = EXOME
• Often we are only interested in gene protein coding exons
• Exome sequencing is more efficient than whole genome sequencing and is more cost effective

Question 10

How is exome sequencing carried out?

Answer 10

Carried out by target enrichment - you want to enrich the DNA library with just exons

• You would hybridise the library with baits made of RNA,
• The baits will be complementary to the desired exons
• Magnetic beads will pull the fragments out

Question 11

What is the difference between NGS vs Sanger Sequencing?

Answer 11

• NGS will produce a digital readout (left) vs Sanger will produce an analogue read out (right)
• Sanger is one sequence read, NGS is a consensus sequence of many reads

Question 12

What are the main drives for this research?

Answer 12

• You are able to explore genetic diseases with NGS
• Use NGS to identify what disease causing gene they may have
• e.g perform exome sequencing and compare variant profiles of affected individuals and look for mutations

Diagram shows patient DNA sample which has been subjected to exome sequencing, reveals heterozygous mutation in the CFTR gene

Question 13

Describe RNA sequencing

Answer 13

NGS can also be used for RNA seq

1. RNA is first converted to cDNA prior to library construction via reverse transcriptase
2. After this the same method is followed for DNA library fragmentation and sequence construction

• NGS of RNA samples determines which genes are actively expressed.

A single experiment can capture the expression levels of thousands of genes

• The number of sequencing reads produced from each gene can be used as a measure of gene abundance
• The amount of sequencing you have for each gene is proportionate to its expression level which allows you to calculate the differences in gene expression of all genes in the experimental conditions
• With appropriate analysis RNA seq can be used to discover distinct isoforms of genes which are differentially regulated and expressed

Question 14

Describe third generation sequencing

Answer 14

Also known as Oxford, Nanopore Sequencing

DNA is forced through a nanopore, this generates an electrical signal which gives rise to the sequence