Lecture 22 - Genomics and Bacterial Evolution Flashcards Preview

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Flashcards in Lecture 22 - Genomics and Bacterial Evolution Deck (50):
1

Functional genetics

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

2

Study of a single genome

Genomic analysis

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Study of several genomes

Comparative genomics

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What did Fred Sanger initially sequence?

PhiX bacteriophage

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Size of PhiX

~5Kb

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Sanger sequencing method
1)
2)
3)
4)

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

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Read size of sanger sequencing

~600bp

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Read

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

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Read assembly

Reads are placed together, according to consensus sequences.
This forms a contig, which is a sequence of reads

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Contig

Where read sequences overlap, make a sequence of consensus sequences

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Gap

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

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Why can gaps occur?
1)
2)

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.

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Automated Sanger sequencing method

Capillary electrophoresis

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

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.

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Key difference between Sanger and Illumina

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

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MiSeq output per run

15Gb

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NextSeq500 output per run

120Gb

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HiSeq2500 output per run

1000Gb

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MiSeq read number

25 million

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NextSeq500 read number

400 million

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HiSeq2500 read number

4000 million

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MiSeq read length

2x300bp

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NextSeq500 read length

2x150bp

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HiSeq2500 read length

2x125bp

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MiSeq time for run

~4 hours

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Most inexpensive sequencing method

Illumina

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PacificBio RS output per run

375Mb

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PacificBio RS read number

~45,000

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PacificBio RS read length

Over 20Kb

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What is PacificBio RS?
1)
2)
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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

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

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

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Size of wells used in PacificBio RS

Zeptolitre quantities

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

Not all DNA is amplified at equal levels.
This can affect results

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What are long read lengths useful for?

For complex sequences of DNA, such as repeat regions.

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Sanger output per run

9600bp

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Read number of sanger

96

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Sanger run time

3 hours

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PacificBio RS run time

30 minutes - 3 hours

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Sanger cost per Mb

$2400

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Illumina cost per Mb

$0.15

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PacificBio RS cost per Mb

$1

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Genome annotation

A process which locates genes in a genome map

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How to annotate a genome
1)
2)

1) Identify open reading frames
2) Experimentally identify gene function, or compare to other genes

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Open reading frame

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

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Bioinformatics
1)
2)
3)

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

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Significance of an open reading frame

Presumed to encode a protein

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BLAST

Basic local alignment search tool

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A tool used in bioinformatics

BLAST

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BLAST role

Compares primary sequence information from different genomes

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Type of sequencing methods that Illumina and PacificBio RS are

Sequencing by synthesis