Lecture 22 - Genomics and Bacterial Evolution

Question 1

Functional genetics

Answer 1

Work out how a protein works from the genetic code, and experimental data.

Question 2

Study of a single genome

Answer 2

Genomic analysis

Question 3

Study of several genomes

Answer 3

Comparative genomics

Question 4

What did Fred Sanger initially sequence?

Answer 4

PhiX bacteriophage

Question 5

Size of PhiX

Answer 5

~5Kb

Question 6

Sanger sequencing method
1)
2)
3)
4)

Answer 6

1) Break sequence of interest into fragments
2) Place in test tube with dideoxynucleotides, each with an individual dye. ddnucleotides terminate chain elongation
3) Run fragments on a polyacrylomide gel, which can resolve to individual base pair level
4) The dye colour of nucleotides is read

Question 7

Read size of sanger sequencing

Answer 7

~600bp

Question 8

Read

Answer 8

The length of a single piece of DNA that can be sequenced by a particular method

Question 9

Read assembly

Answer 9

Reads are placed together, according to consensus sequences.

This forms a contig, which is a sequence of reads

Question 10

Contig

Answer 10

Where read sequences overlap, make a sequence of consensus sequences

Question 11

Gap

Answer 11

When a computer can’t find a match in reads to make a contig

Question 12

Why can gaps occur?
1)
2)

Answer 12

1) DNA polymerase can’t extend sequence for some reason

2) If there is a repeated region, and the read size is smaller than the size of the repeat.

Question 13

Automated Sanger sequencing method

Answer 13

Capillary electrophoresis

Question 14

Illumina sequencing 
1)
2)
3)
4)
5)
6)
7)
8)
9)

Answer 14

1) Break DNA of interest into fragments
2) Adaptors of known sequence are added, ligate to the ends of dsDNA
3) A glass slide is prepared, with sequences complementary to primers adhered to surface
4) Hybridisation of primers, adhered complementary sequences
5) Add unlabelled nucleotides, DNA polymerase. Bridge amplificaiton
6) DNA synthesis, bridges become double stranded
7) Denaturation, to ssDNA
8) PCR to make high-density DNA clusters
9) Bases tagged with fluorescent dyes added. When a base is added, emits fluorescence which is detected.

Question 15

Key difference between Sanger and Illumina

Answer 15

Illumina sequencing can continue on same strand after dye-tagged base is added.

Fluorescent part is cleaved off when base is incorporated, so it doesn’t interfere with further elongation

Question 16

MiSeq output per run

Answer 16

15Gb

Question 17

NextSeq500 output per run

Answer 17

120Gb

Question 18

HiSeq2500 output per run

Answer 18

1000Gb

Question 19

MiSeq read number

Answer 19

25 million

Question 20

NextSeq500 read number

Answer 20

400 million

Question 21

HiSeq2500 read number

Answer 21

4000 million

Question 22

MiSeq read length

Answer 22

2x300bp

Question 23

NextSeq500 read length

Answer 23

2x150bp

Question 24

HiSeq2500 read length

Answer 24

2x125bp

Question 25

MiSeq time for run

Answer 25

~4 hours

Question 26

Most inexpensive sequencing method

Answer 26

Illumina

Question 27

PacificBio RS output per run

Answer 27

375Mb

Question 28

PacificBio RS read number

Answer 28

~45,000

Question 29

PacificBio RS read length

Answer 29

Over 20Kb

Question 30

What is PacificBio RS?
1)
2)
3)
4)
5)

Answer 30

1) Single molecule, real time sequencing
2) DNA synthesis by immobilised DNA polymerase
3) Phospholinked nucleotides release light when incorporated
4) No amplification
5) Under 180 minutes per run

Question 31

PacificBio RS method
1)
2)
3)
4)
5)

Answer 31

1) Don’t fragment DNA of interest too much (reduces read length)
2) Repair ends
3) Adaptor ligation to DNA ends
4) DNA is polymerised by DNA polymerase fixed in a 0-mode waveguide well
5) When a phosphonucleotide is incorporated, light is emitted and detected. Each base has a different dye, and emits a different wavelength of light

Question 32

Size of wells used in PacificBio RS

Answer 32

Zeptolitre quantities

Question 33

Why is it better to not have an amplification stage in sequencing?

Answer 33

Not all DNA is amplified at equal levels.

This can affect results

Question 34

What are long read lengths useful for?

Answer 34

For complex sequences of DNA, such as repeat regions.

Question 35

Sanger output per run

Answer 35

9600bp

Question 36

Read number of sanger

Answer 36

96

Question 37

Sanger run time

Answer 37

3 hours

Question 38

PacificBio RS run time

Answer 38

30 minutes - 3 hours

Question 39

Sanger cost per Mb

Answer 39

$2400

Question 40

Illumina cost per Mb

Answer 40

$0.15

Question 41

PacificBio RS cost per Mb

Answer 41

$1

Question 42

Genome annotation

Answer 42

A process which locates genes in a genome map

Question 43

How to annotate a genome
1)
2)

Answer 43

1) Identify open reading frames

2) Experimentally identify gene function, or compare to other genes

Question 44

Open reading frame

Answer 44

Over 100 codons that are uninterrupted by a stop codon.

See if there is an obvious ribosomal binding site at the 5’ end, terminator sequence at 3’ end

Question 45

Bioinformatics
1)
2)
3)

Answer 45

1) Analysis of a genome using computers
2) Generates information of genome structure, content, arrangement
3) Uses annotation to determine location of genes on newly-sequenced genome

Question 46

Significance of an open reading frame

Answer 46

Presumed to encode a protein

Question 47

BLAST

Answer 47

Basic local alignment search tool

Question 48

A tool used in bioinformatics

Answer 48

BLAST

Question 49

BLAST role

Answer 49

Compares primary sequence information from different genomes

Question 50

Type of sequencing methods that Illumina and PacificBio RS are

Answer 50

Sequencing by synthesis