Genome sequencing and assembly 2

Question 1

Better accuracy out of sanger and NGS?

Answer 1

Sanger

Question 2

Cost and time effectiveness for sanger vs NGS?

Answer 2

Less than 20 samples = sanger
More than 20 samples = NGS

Question 3

Sample coverage for sanger?

Answer 3

1read/sample

Question 4

Sample coverage for NGS?

Answer 4

billions of reads per sample

Question 5

Main benefit of oxford nanopore sequencing?

Answer 5

Portability

Question 6

Example of a use of oxford nanopore?

Answer 6

Could sequence viral outbreaks in the field

Question 7

Main component of oxford nanopore?

Answer 7

Nanopore embedded in an artificial membrane

Question 8

What happens to the DNA in oxford nanopore?

Answer 8

ssDNA is passed through the nanopore

Question 9

What is measured in oxford nanopore?

Answer 9

Current across the membrane

Question 10

How are the nucleotides determined in oxford nanopore?

Answer 10

Each base affects the current going through the nanopore in their own way, and these changes are recorded

Question 11

WHat unwinds the DNA in ON?

Answer 11

Motor protein

Question 12

Roles of motor protein in ON?

Answer 12

Guides the DNA and unwinds it

Question 13

What is unique ab ON?

Answer 13

Generates sequencing data in real time

Question 14

Strengths of ON?

Answer 14

Can do v long reads (>10,000bp)
Can read DNA & RNA

Question 15

Weaknesses of ON?

Answer 15

High error rate–> 1-15%

Question 16

Main components of PacBio sequencing?

Answer 16

DNA prep, loading, sequencing

Question 17

Prep part of PacBio sequencing?

Answer 17

DNA is fragmented and tagged w/ special adaptors that create circular DNA molecules

Question 18

Loading part of PacBio sequencing?

Answer 18

DNA is put into sequencing chambers called zero mode wave guides

Question 19

What are zero mode wave guides?

Answer 19

Microscopic sequencing chambers
Each well has DNA pol which synthesises the new strand by adding fluorescently labelled nucleotides

Question 20

Sequencing/recording in PacBio sequencing?

Answer 20

Each base emits fluorescence as it is being incorporated into the strand

Question 21

Benefits of PacBio sequencing?

Answer 21

V accurate

Question 22

Limitations of PacBio sequencing?

Answer 22

V expensive

Question 23

What is shotgun sequencing?

Answer 23

Randomly breaking up DNA into small fragments, sequencing and reassembling

Question 24

Use of shotgun?

Answer 24

Large genome sequencing

Question 25

Steps of shotgun?

Answer 25

Copying, Fragmentation, sequencing, computational assembly

Question 26

What is genome assembly?

Answer 26

The process of reconstructing a genome from short or long read sequencing reads

Question 27

Two types of genome assembly?

Answer 27

De novo assembly and Reference-guided assembly

Question 28

De novo assembly?

Answer 28

Assembling a genome from scratch without a reference

Question 29

Reference guided assembly?

Answer 29

Using a known genome as a scaffold

Question 30

First genome to be shotgun sequenced?

Answer 30

Influenza