Genome sequencing and assembly 1

Question 1

What is genome sequencing?

Answer 1

Process of determining the complete DNA sequence of an organisms genome

Question 2

Importance of genome sequencing?

Answer 2

Helps understand genetic info, evolutionary biology and medical research

Question 3

What is a genome?

Answer 3

The complete set of genes or genetic material present in an organism

Question 4

Three types of genome sequencing?

Answer 4

Sanger sequencing, next gen sequencing and third gen sequencing

Question 5

Other name for sanger sequencing?

Answer 5

Chain termination method

Question 6

First step of sanger sequencing?

Answer 6

Denature DNA strand to get antisense strand

Question 7

Second step of sanger sequencing?

Answer 7

Add radioactively labelled primers to the DNA
Add nucleotides and polymerase

Question 8

What is done to the DNA in sanger sequencing after the radioactively labelled primers were added

Answer 8

Sequencing was randomly stopped at specific nucleotides–> i.e. could get it to stop at an A, C, T, G

Question 9

What was done after the fragments were generated in sanger sequencing?

Answer 9

Ran on a gel–> shorter fragments ran longer

Question 10

What was done after running the fragments on a gel in sanger sequencing?

Answer 10

put xray film over them–> could see what the end nucleotide was as it was the one that was radioactively labelled

Question 11

How was the DNA extension stopped at specific nucleotides in sanger sequencing?

Answer 11

Used a modified nucleotide that didn’t have the 3’ OH group–>chain synthesis was terminated

Question 12

What are the modified nucleotides used in sanger sequencing called?

Answer 12

Dideoxynucleotides

Question 13

Issue with using radioactively labelled primers in sanger sequencing?

Answer 13

X-ray took a while to develop

Question 14

What was used instead of radioactively labelled primers?

Answer 14

Used fluorescent dideoxynucleotides–> each base was a diff colour

Question 15

Benefit of using fluorescent dideoxynucleotides in sanger sequencing?

Answer 15

Can have an instant read off the gel instead of having to do an x ray
Can run the reaction on one lane

Question 16

What was used instead of gel in sanger sequencing?

Answer 16

Capillary tubes,

Question 17

How were capillary tubes used in sanger sequencing?

Answer 17

pass a fluorescence detecter

Question 18

Benefit of using a capillary tube in sanger?

Answer 18

COuld be automated–> multiple tubes could run at the same time in parallel

Question 19

How long does it take 1 sanger machine to sequence a human genome?

Answer 19

1.2yrs

Question 20

Limitations of sanger?

Answer 20

Time consuming and expensive for large genomes

Question 21

APplications of sanger?

Answer 21

Still used for small genomes or targeted sequencing–> individual genes

Question 22

Other names for next gen sequencing?

Answer 22

Short read sequencing ,illumina sequencing

Question 23

What is PCR used for?

Answer 23

Amplification of DNA sequences

Question 24

PCR process?

Answer 24

Denature DNA–>92 degrees
Primers added and anneal to the DNA–> 50-68
Extension–> nucleotides and DNA pol added–> 72 degrees

Question 25

Main steps in illumina sequencing?

Answer 25

Sample prep Cluster amplificaiton Sequencing by synthesis

Question 26

Sample prep in illumina?

Answer 26

Fragment DNA into 200-500bp pieces, ass adaptors to the ends of the fragments

Question 27

CLuster amplification in illumina?

Answer 27

DNA fragments bind to flow cell via complementary adaptors Amplification creases clusters of identical DNA molecules

Question 28

How can DNA be fragmented for ilumina?

Answer 28

Sonication--> high frequency sound waves

Question 29

Why are adaptors added in illumina?

Answer 29

Allow DNA to attach to the flow cell and act as primers

Question 30

General idea of sequencing by synthesis?

Answer 30

Works by sequencing the DNA as it is being synthesised, one base at a time

Question 31

First bit of sequencing by synthesis?

Answer 31

Clusters of identical fragments are formed on the flow cell DNA pol begins synthesising the complementary strands

Question 32

How is it known what nucleotides are being added in sequencing by synthesis?

Answer 32

Nucleotides have a diff fluorescent tag corresponding to a base

Question 33

How does sequencing by synthesis detect which base was added?

Answer 33

As the nucleotide is added to the strand it emits a fluorescent signal

Question 34

Main diff between sanger and ilumina?

Answer 34

In ilumina, after each nucleotide is incorporated and the fluorescence recorded, the terminator bit and the dye is cleaved off so a new nucleotide can be added and recorded

Question 35

Benefits of NGS?

Answer 35

High throughput, cost effective for big projects

Question 36

Limitations of NGS?

Answer 36

Short reads, high computational demands