Genetics

Question 1

What is the basic overview of sanger sequencing?

Answer 1

Prepare a PCR reaction -> capillary gel electrophoresis sequencer -> detection fo DNA fragments -> computer analysis -base calling -> visualisation of sequence trace

Question 2

How does sanger sequencing differ from PCR?

Answer 2

It incorporates the ddNTPs which terminate sequencing at the site of its location

Question 3

What does the Sanger sequencing reaction consist of?

Answer 3

Taq polymerase + buffer + primer + template + ddnucleotides + dnucleotides

Question 4

How is each base differentiated upon analysis?

Answer 4

Each base is assigned a different flourophore

Question 5

Explain the basic principle of sanger sequencing.

Answer 5

Works similar to PCR to amplify segments of DNA. In sanger the target DNA is replicated, but cut at random points, due to the incorporation of ddNTPs.
Results in the synthesis of many fluorescently labelled oligonucleotides

Question 6

What errors may occur during sanger sequencing?

Answer 6

Poor quality template
Contaminants in your reaction
Secondary structure in DNA
Poor primer design

Question 7

What is next generation sequencing?

Answer 7

Utilises high-throughput, parallel approach to sequencing large numbers of different DNA sequences in a single reaction.

The technology is based on the detection of specific bases as they are added to the complementary strand rather than termination of elongation

Question 8

Which sequencing technique is best described by sequencing by synthesis?

Answer 8

Next generation sequencing

Question 9

Compare sanger and NGS.

Answer 9

Sanger is more accurate, but NGS can investigate more than 100 genes at once.
Sanger is more appropriate for long reads whilst NGS is better for novel variants in many targets.

NGS can be utilised in whole genome studies or genome wide association studies.

Interestinlgy Sanger is still the gold and can be used to confirm a NGS

Question 10

State the overview of NGS

Answer 10

1. Fragment gDNA
2. Ligate adapters
3. Cluster generation on flow cell
4. Sequencing

Question 11

Describe the process of NGS

Answer 11

1. Fragment gDNA - to a uniform size. Requires size distribution confirmed by an agarose gel
2. Ligate adapters (sequence enriching) - Used to produce a sequence library. Adapters are ligated to both ends of the DNA to allow pair density sequencing and allow fragment to bind to flow cell. Each individual is barcoded
3. Cluster generation on flow cell by bridge amplificaiton to produce clusters of the same seqence, known as clonal libraries
4. Sequencing - by synthesis technology is used to detect each base incorporate into the DNA fragment by the addition of fluorescently labelled nucleotides

Question 12

What is the first step to NGS?

Answer 12

Sample prep

Question 13

What does sample prep in NGS involve?

Answer 13

DNA fragmentation (enzymatic or physically), end repair, phosphorylation of the 5 prime end, A-tailing of the 3 ends, ligation of oligonucleotides adapters, some PCR cycles to enrich for product that has adapters ligated to both ends.

Question 14

In illumina sequencing what is the aim of library preparation?

Answer 14

Aim of the library is to obtain NA fragments with adapters attached on both ends (allow cluster generation)

• Index: allows sample barcoding
• Sequencing primers x2

Question 15

What is an exception to the NGS sample prep steps?

Answer 15

Illuminas Nextera XT

• Single tube reaction termed tagmentation
  
  • Combines fragmentation, end-repair and adapter ligation in a single step

Question 16

Describe cluster generation in illumina sequencing?

Answer 16

• Cluster generation occurs on a flow cell
• Flow cell is a thick glass slide with channels or lanes
• Each lane is randomly coated with a lawn of oligos that are complementary to library adapters

Question 17

How is cluster generation/amplification achieved in Illumina sequencing?

Answer 17

Nucleotides are added along with a polymerase to extend bound libraries. The extension occurs on complementation with the lawn of oligos. Extension occurs from the 3 prime end.

The newly synthesised strand is complemtary to the template. It is then denatured to wash away the original template. The newly synthesised strand is covalently attached to flow cell surface

Question 18

What is bridge amplification?

Answer 18

The newly synthesised strand flips over and forms a bridge by hybridising to adjacent complementary primer.

Hybridised primer is extended by polymerase forming a double stranded bridge

Question 19

What is the template DNA in NGS?

Answer 19

This is the DNA fragments from the sample prep, which have primers ligated onto them with complementation to the oligo lawn

Question 20

Why are there 2 different types of oligos on the flow cell?

Answer 20

This allows for bridge amplification. Where the oligo at one end of the template will complement to the attached oligo, whilst the other oligo will allow complementation to the neighboring oligo - forming a bridge

https://www.youtube.com/watch?v=fCd6B5HRaZ8&ab_channel=Illumina

Question 21

What follows the final bridge amplification?

Answer 21

The dsDNA bridges are denatured to form linear forms of single stranded DNA tethered to the oligo lawn.

Reverse strands are washed away to leave the ones identical to the original template left.

The 3 prime ends are block from excess oligos

Question 22

What is the process of sequencing by synthesis?

Answer 22

Each nucleotide added has a distinct fluorescent label. It has a reversible blocking R-group at the 3´ carbon of the sugar molecule instead of OH.
- The base molecule is bound via a cleavable linker to the fluorophore - one for each base

Because of blocker group strand extension stops upon the addition of modified nucleotide containing blocker. Incorporated nucleotide is then removed and fluorescent signal is detected to ID ATG or C. The nucleotide is regenerated by adding the OH meaning it can add back normally to continue this form of fluoresence.

Question 23

Define depth in NGS data analysis?

Answer 23

Number of times a nucleotide is read during the sequencing process

Question 24

Define coverage in NGS data analysis?

Answer 24

Basically how many times one part of a fragment has been read. Done using overlaps

Question 25

What are some data analysis issues associated with NGS?

Answer 25

The sheer volume of data produced causes problems with processing times and storage.

Network traffic needs to be prioritised in hospital.

Question 26

Clinics challenges for NGS

Answer 26

Cost:benefit, sample origin/quality, resolution, reproducibility, reporting standards, storage, ethics, biological interpretation

Question 27

What is 3rd generation sequencing?

Answer 27

The ability to sequence long strands of DNA with little preparation using nanopores.

Can deliver up to 1000bases

Currently moving into clinic and public health for quick real time applications for looking at RNA expression and also structural complexitites

Question 28

What does the first read sequence?

Answer 28

The forward strand

Question 29

What does the second read sequence?

Answer 29

The reverse strand

Question 30

How does the first read in NGS commence?

Answer 30

Begins with the binding of the template to oligo lawn. The template is read by DNA polymerase to produce a complementary strand. That complementary strand is then used to further bridge amplify and produce a copy of the template strand. Once complete the reverse strands are washed and the forward strands are 3´ blocked to begin sequencing.

Question 31

What occurs following the first read?

Answer 31

The read product is washed away and the 3´ block is removed. Index 1 read primer is introduced to allow for barcoding. The template is now able to bridge amplify again. Following dsDNA the two are linearised and the template is removed. The 3´ end is blocked and sequencing begins

Question 32

How is ambiguous alignment produced?

Answer 32

By using pair-end information

Question 33

How is the length of read indicated in NGS?

Answer 33

Number of cycles = length of read