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Chaudhari Flashcards

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1
Q

give 2 examples of first generation sequencing and whether they are used or not

A 
maxam gilbert (not used)
Sanger (used)
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2
Q

give 2 examples of second generation sequencing technologies

A

illumina

ion torrent

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3
Q

give 2 examples of 3rd gen sequencing technologies

A

pacific biosciences

oxford nanopore

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4
Q

what is sanger good at sequencing

A

low volume targetted sequences (not good for genome sequencing but useful for smaller reads)

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5
Q

what kind of reads does illumina produce

A

many short (50-300bp) reads

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6
Q

what kind of reads do oxford nanopore generate compared to illumina

A

fewer reads but very long reads (>10kb)

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7
Q

what are the 3 `advantages of second gen sequencing (illumina) over sanger

A

massively parallel - a single gene run generates millions of sequences
much cheaper per base
built in shot gun sequencing without a cloning step

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8
Q

what are the disadvantages of second gen sequencing technologies compared to sanger

A

library prep is expensive and slow
amplification of DNA fragments is required which can introduce biases
read lengths are quite short

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9
Q

what is good about third gen

A

good for finished gnomes (give n gaps) and base modifications (epigenetics)

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10
Q

individual reads in third gen have a quite high error rate T/F

A

T

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11
Q

what are the applications of illumina

A

draft genome sequencing,
resequencing
functional genomics

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12
Q

what are the applications of Pac bio

A

complete genome sequencing,

detection of DNA methylation

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13
Q

what are the applications of oxford nanopore

A

complete genome sequencing,
epigenetics
direct RNA-seq
metagenomics

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14
Q

what are the key steps to illumina sequencing

A
  1. extract genomic DNA
  2. fragment genomic DNA
  3. add linkers
  4. add input library to flow cell
  5. Amplification (bridge amplification)
  6. sequencing
  7. image taken
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15
Q

each flow cell cluster corresponds to what

A

a separate read

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16
Q

patterned flow cells make the system less prone to what

A

overclustering

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17
Q

what is a patterned flow cell

A

there are individual nanowells tha contain the primers for bridge amplification. A cluster is generated within each well and cant spread outside the well so overlapping clusters do not form

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18
Q

what is meant by phasing when referring to illumina

A

In sequencing-by-synthesis chemistry like Illumina (sorry, Solexa!) phasing is the rate at which single molecules within a cluster loose sync with each other. Phasing is falling behind, pre-phasing is going ahead and together they describe how well the chemistry is performing

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19
Q

once you get above 100 bases in illumina there is a lot of confusion and phasing. this is why illumina reads are limited to being short T/F

A

T

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20
Q

phasing problems result in a reduction of quality towards the end of reads T/F

A

T

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21
Q

what is 2 colour illumina sequencing

A 
4 bases are sequenced using only 2 colours eg red and green
1st base - green
2nd base - red
3rd base - both
4th base -non
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22
Q

what are the benefits of 2 colour illumina sequencing

A

it allows simplified optics so has lower costs

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23
Q

how do oxford nanopore sequence

A

a few bases are inserted into the pore, there is a change in electrical potential and the sequence is determined

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24
Q

what is the highly portable version of the oxford nanopore known as

A

minION

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25
Q

what is the promethION

A

essentially lots of minIONs in a suitcase

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26
Q

what is GridION

A

somewhere in between minION and promethION - 5 minION flow cells

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27
Q

when was the first bacterial genome sequence published

A

1995

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28
Q

what was the first bacterial genome sequencing project to be INITIATED

A

E.coli K-12

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29
Q

which were the first bacterial genomes to be sequenced and how was this done

A

Haemophilus influenzae and mycoplasma genitalium

shotgun sequencing

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30
Q

what does shotgun sequencing rely on

A

computational assembly of sequence from random clone libraries, randomly sequence part of the genome and then place together in a contig sequence

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31
Q

what is de novo assembly

A

process of merging overlapping

sequence reads into contiguous sequences (contigs) without the use of any reference genome as a guide

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32
Q

what is the best reference to use to order contigs

A

usually the most closely related bacterium with a ‘finished’ genome

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33
Q

Due to evolutionary differences between the reference
and novel genome, the presence of (often mobile) repeat elements such as prophages, and the very nature of short-read assemblers, there will almost certainly be assembly errors present within the contigs.

A

T

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34
Q

Once the ordered set of contigs has been obtained, what is the next step

A

to annotate the draft genome -the process of ‘gene’ finding

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35
Q

Multi-locus sequence typing (MLST) is a widely used,

sequence-based method for typing of bacterial species and plasmids T/F

A

T

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36
Q

what are gaps in the draft genome from shotgun sequencing due to

A
  • repeats
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37
Q

how can you fill in the gaps in a draft genome

A

PCR from the contigs

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38
Q

how were genes that are toxic to bacteria identified

A

Looked at data from previous experiments for multiple microbial genomes that used sanger in clone by clone approach
The gaps in this sequence must have been there for a reason (if clone contained that bit of genome may be toxic to cell your growing plasmid up in)
Identified compounds from these gaps that were toxic to e.coli

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39
Q

where was e.coli k12 originally isolated from

A

a convalescent diptheria patient in 1922 (part of the guts flora)

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40
Q

how many protein coding genes does e.coli k12 have

A

4288

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41
Q

how were regions of low GC content acquired in e.coli k12

A

horizontal transfer

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42
Q

A girl in the isle of white became infected by which strain of e.coli

A

O157:H7
an emergent human pathogen associated with haemorrhagic colitis (blood in diahorrea) and haemolytic uraemic syndrome (HUS), which can lead to kidney failure and is sometimes fatal

43
Q

how much bigger is the genome of O157 compared to K12

A

1mb

44
Q

when was the genome of O157 sequenced

A

2001

45
Q

what is CFT073

A

a strain of uropathogenic E.coli (UPEC)

not as dangerous as O157

46
Q

what is CFT073 an example of

A

a extraintestinal E.coli (ExPEC) - associated with UTIs

47
Q

ExPEC can be harmless when in the intestines but become pathogens when they invade where

A

urinary tract, blood or cerebrospinal fluid

48
Q

the genome of CFT073 is similar in size to O157 but the extra sequences relative to K12 are not the same as O157

A

T

49
Q

how big is the core genome between K12, O157 and CFT073

A

3000 genes

50
Q

how do you estimate the size of the E.coli core genome

A

randomise the order in which you sequence strains. count the number of conserved genes
the number of conserved genes decreases as the amount of strains increases

51
Q

how big is the core genome of E.coli expected to be

A

around 2229 genes

52
Q

how do you predict the pangenome of E.coli

A

sequence strains and count the number of genes

The e.coli genome is effectively infinite - you will always find new gene

53
Q

when looking for the number of unique genes across E.coli what was found

A

there will always be 300 genes that you havent sequenced before

54
Q

sequencing to the draft stage is far cheaper than completion

A

T

55
Q

what did the GWAS study find about host specificity in campylobacter

A

genes involved in B5 biosynthesis are present in strains that infect cows but not chickens

56
Q

who discovered E.coli

A

Theodor Escherich

57
Q

who discovered shigella

A

kiyoshi shiga

58
Q

e.coli and shigella are distinguished on the basis of what?

A

motility, metabolic profile and clinical manifetation

59
Q

E.coli will grow on lactose whereas shigella would not

A

T

60
Q

E.coli are usually commensal whereas shigella are what?

A

obligate pathogens

61
Q

what is the process of serotyping

A

• Taking a particular antigen and raising antibodies against it
• See if these cross react with antigen from other strains
• If they cross reaction- then those are of the same serotype
– Typing based on immune recognition of cell surface antigens

62
Q

how were strains distinguished after serotyping

A

cross hybridisation

63
Q

• Milkman (1973) began the quantitative study of E. coli population genetics by measuring the electrophoretic mobility of enzymes derived from different E. coli strains. this was known as…

A

multi locus enzyme electrophoresis (MLEE)

64
Q

what is the ECOR collection

A

• A set of 72 phylogenetically diverse E.coli strains, chosen based on MLEE data

65
Q

shigella has arisen on multiple occasions from E.coli, how do we know

A

Shigella is split up into different serotypes, these are split up within in the E.coli phylogenetic tree

66
Q

what does multi locus sequence typing (MLST) do

A

involvesobtaining the nucleotide sequences of 450 bp fragments derivedfrom (typically) 6–8 housekeeping genes at distinct loci around thebacterial chromosome.
ince they are likely to be under strong purifying selection, and theobserved variations are likely to be selectively neutral.

67
Q

what was the first application of MLST

A

investigated the relationship between a number of EPEC and EHEC strains by analysing 7 house keeping genes

68
Q

the radiation of clones began about 9 million years ago
and the highly virulent pathogen responsible for epidemics of
food poisoning, E. coli O157:H7, separated from a common
ancestor of E. coli K-12 as long as …. years ago

A

4.5 million

69
Q

Phylogenetic analysis reveals that old lineages of E. coli have acquired the
same virulence factors in parallel, including a pathogenicity
island involved in intestinal adhesion, a plasmid-borne haemolysin, and phage-encoded Shiga toxins. T/F

A

T

70
Q

what type of phylogenetics is the gold standard

A

core genome phylogenetics

-phylogeny based ont the core genome

71
Q

what is 16s ribosomal RNA profiling good for investigating

A

diversity of organisms ACROSS different species

72
Q

16S ribosomal RNA is present in all bacteria and archaea T/F

A

T

73
Q

16S ribosomal RNA has both constant and variable regions. how are these used for its analysis

A

constant regions are used as primer binding sites. V loops can be used to determine evolutionary relationships between strains

74
Q

what are the caveats of 16S rRNA profiling

A
  • primers may not be truly universal
  • contamination can be an issue
  • sequencing errors can result in the overestimation of diversity of organisms present
  • some organsims have multiple copies of 16S rRNA genes which vary in sequence (can result in overestimation of taxa present)
  • PCR bias may result in incorrect quantification of species
75
Q

the first real application of 16S rRNA to understand the diversity was seen in what

A

Carl Woese 3 domain structure of life

76
Q

what is meant by microbial “dark matter”

A

up to 99% of bacteria we cannot culture

77
Q

what is metagenomic

A

the study of genetic material recovered directly from environmental samples

78
Q

To comprehensively sample an environment what is required

A

deep sequencing

79
Q

individual sequences can be derived from metagenomic studies how

A

using software such as Kraken
de novo assembly can be used to piece together larger contigs from whole bacterial genomes. This is an incredibly difficult computational problem
However, assembly of metagenomic data is complex, computationally intensive and error prone

80
Q

what was found to critically impact microbiome analysis

A

reagent and laboratory contamination

81
Q

the reagents used for DNA extraction are routinely contaminated with microbial DNA sequence T/F

A

T

82
Q

A paper reported anthrax in samples taken from the subway. what was wrong with this conclusion.

A

sequenced the sample and identified the most common hit. pathogenic and non pathogenic strains are very similar and the proper toxins were not found to be present so it wasnt proper anthrax

83
Q

in single cell genomics how can individual cells be isolated

A

optical tweezer, cell sorting FACs, micropipetting or laser microdissection

84
Q

how is the genome in single cells processed before sequencing

A

single copy of the genome is PCR amplified, sequenced and assembled

85
Q

what is iChip a method of

A

culturing bacteria so that there is enough material for DNA sequencing

86
Q

how is iChip carried out

A
  • Samples are diluted so on average one bacterial cell ends up in each chamber in the iChip
  • The chambers are filled with molten agar and covered with a semi-permeable membrane
  • The iChip is placed back in the native environment
  • This allows a colony to grow from a single cell, which provides enough material for DNA sequencing
87
Q

what was the new antibiotic identified by iChip called

A

Teixobactin

88
Q

early studies used what to investigate bacterial diversity

A

serotyping, DNA-DNA hybrisation and MLEE

89
Q

sequencing of what is useful for identifying the bacterial species within a sample

A

16S ribosomal RNA

90
Q

metagenomics, single-cell sequencing and iChip allow additional sequence data to be obtained from unculturabel micoorganisms T/F

A

T

91
Q

what was the first pathogenic E.coli to be characterised

A

enteropathogenic E.col (EPEC)

92
Q

what type of secretion system does EPEC encode

A

Type III - which it used to inject host cells with effector molecules, allowing it to attach to the gut wall

93
Q

whats the difference between EPEC and EHEC

A

EHEC encode a shiga tocin which is associated with more severe disease

94
Q

what is an example of an EHEC

A

E.coli O157:H7

95
Q

what are enteroaggregative E.coli (EAEC)

A

mild pathogens associated with diarrhoea that stick together in a characteristic stacked brick conformation to aid survival in the gut

96
Q

what country did the e.coli outbreak in 2011 occur in that mainly affected young women

A

germany

97
Q

what did the young women in germany in 2011 mainly suffer from

A

haemolytic uremic syndrome

98
Q

what did the find when they looked for the commonalities of all the young women in germany affected by E.coli strain

A

they all had ate cucumbers recently

99
Q

what mistake did german health authorities make

A

linked O104 serotype to cucumbers imported from spain

was actually serotype O157:H7 originated from beansprouts

100
Q

what did genomic sequencing by BGI confirm

A

O104:H4 serotype has some enteroaggregative E. coli (EAEC or EAggEC) properties, presumably acquired by horizontal gene transfer

101
Q

what caused the outbreak in germany and surrounding countries in 2011

A

strain of E. coli of the serotype O104:H4, that was unusual for having characteristics of both enteroaggregative E. coli and enterohemorrhagic E. coli. The strain has a number of virulence genes typical of enteroaggregative E. coli, including attA, aggR, aap, aggA, and aggC, in addition to the Shiga toxin variant 2

102
Q

all sequence data from the german 2011 outbreak was available during the course of the outbreak T/F

A

T

103
Q

the two outbreak strains in germany 2011 were identical to what at the core genome level showing it was an enteroaggregative e.coli

A

55989

104
Q

PacBio sequencing of the O104:H4 was not possible during the outbreak T/F

A

T

Genomic Science (2 decks)

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