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BS22002 > Genomics > Flashcards

Genomics Flashcards

1
Q

Dye determination sequencing

A
  • each ddNTP is labelled with a different fluorophore

- one reaction with all four ddNTPS

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2
Q

Single sequence to genome

A
  • genome too long to sequence in one go
  • can fragment the genome into large pieces (50-200kpb) which can be amplified in bacterial culture as bacterial artificial chromosomes (BACs).
  • similar strategies in yeast and cosmids/fosmids which use other replication origins in bacteria
  • fragments of DNA are cloned into the vector of choice and then tens of thousands of individual colonies are picked to create a library
  • each clone contains a (hopefully) unique fragment of the genome sequence
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3
Q

BAC library

A
  • rare cutting enzyme digest and clone (every 50-200kb)
  • clone into a BAC vector to give bacterial artificial chromosomes
  • pick each clone into a separate well - each well now contains a different genome fragment
  • each fragment can be purified and analysed
  • align by digest ‘fingerprint’
  • shotgun sequence each BAC individually
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4
Q

Individual BAC clones

A
  • have a restriction digest pattern matching that of the original genome
  • by digesting with many enzymes the digestion patterns can be determined and matched to ‘tile’ BAC clones to give a physical map
  • may also contain known DNA sequences or markers which can then be used to improve the physical map and link it to the genetic map
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5
Q

‘Shotgun’ sequencing with Sanger

A
  • analyses all the bases at once for a single sequence
  • breaks down the genome into manageable chunks at random then sequence
  • fragment genomic DNA -> clone into sequencing vector -> pick colonies and sequence
  • large libraries with tens of thousands of these clones can be constructed and mapped by restriction mapping
  • requires the source DNA to be broken into approx. 1000bp chunks
  • these are incorporated into a sequencing vector and sequenced using standard primers from both sides
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6
Q

Overlap Layout Consensus Method

A
  • DNA is sequenced to produce a set of partial sequences (reads)
  • a computer is used to assemble the sequence reads into a series of overlapping fragments
  • the overlaps are removed by the computer to produce a single assembled sequence
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7
Q

‘Next generation’ sequencing

A

Sanger

  • shotgun cloning is slow and expensive
  • sequences one molecule at a time
  • accurate

Illumina (Solexa)

  • sequences all molecules at the same time
  • quite accurate
  • other competing technologies as well
  • relatively short reads
  • expensive for single sequences, cheap for many
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8
Q

High-throughput sequencing

A

Involves:

  • the chemical amplification of DNA fragments
  • the synthesis of complementary strands using fluorescently labelled nucleotides
  • now outdated and rarely used
  1. single DNA molecule are attached to a solid surface
  2. each molecule is amplified in place by PCR (each spot is a PCR colony or ‘polony’)
  3. the four nucleotides (as nucleotide triphosphates), each labelled with a different fluorescent dye, are added, along with DNA polymerase and a universal primer
  4. only one nucleotide is attached to the primer by DNA polymerase. Unicorporated nucleotides are removed
  5. the newly added nucleotide is detected by a camera
  6. the cycle is repeated about 100 times
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9
Q

High-throughput sequencing

A
  1. The sequence of interest is first fragmented and fragments of a specific size isolated.
  2. Specific PCR primers are ligated onto the ends.
  3. These fragments are then hybridised to oligos on a flow cell (very dilute)
  4. The oligos attached to the flow cell act as primers to amplify the fragment attached to the slide. This forms a PCR colony of identical fragments
  5. then it is on to the sequencing process.
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10
Q

The HTS cycle

A
  1. add to growing chain 5’-3’
  2. detect label with camera
  3. chemically cleave the label revealing the 3’OH
  • limit = about 130-150 cycles
  • further extension blocked by the dye label
  • immobilised template is hybridised with a 3’-labelled dNTP and the sequence extended by one base
  • as 3’ is blocked (the fluorescent label acts as a protecting group), the chain cannot be extended
  • excess reagents are removed and the presence of the fluorescent label is detected
  • the 3’ position is deprotected, ready for the next cycle
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11
Q

Illumina Hiseq

A
  • a small portion of the sequencing slide can read >150 million sequences at the same time for each sample
  • scaling this up, an illumina hiseq has 8 lanes
  • each lane can be used for a different sample
  • each lane can give 20-40 million sequences up to 150 bases
  • 1 run takes 3-6 days (~1 hour per base)
  • 3010^68 lanes * 150 bases = 36 Gbo (approx. ten human genomes)
  • now up to 150Gb per run (50x coverage)
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12
Q

Chromosomes

A

have a single DNA molecule with specialised DNA sequences for the initiation of DNA replication, for spindle interactions in mitosis (centromeres), and for maintaining the integrity of the ends (telomeres)

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13
Q

Protein gene expression

A

occurs at open reading frames, from which RNA polymerase transcribes mRNAs that are translated to form polypeptides, which become functioning proteins. Genes contain DNA sequences for control of their expression

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14
Q

Protein coding genes

A

generally not repetitive but there are some exceptions, e.g. gillagrin and high copy number genes

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15
Q

Repetitive regions

A

microsatellites, telomeres, intron sequences

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16
Q

tRNA

A

very similar sequences (but very short)

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17
Q

rRNA

A

many copies of some ribosomal genes

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18
Q

Transposons

A

mobile genetic elements - sequence of a few kb that can move about the genome. Thousands of copies in eukaryotes

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19
Q

Size matters

A
  • the longest repeats in microbial genomes are about 7kb
  • with the latest technologies we can read right through them
  • without extra long reads we need to improvise with paired-end reads
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20
Q

Contig

A

a contiguous (continuous) consensus sequence from an assembly

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21
Q

Scaffold

A

a series of contigs where we have additional information to place them together in the right order and orientation but the sequence between the contigs is not complete

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22
Q

Assembly

A

the set of scaffolds for one genome

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23
Q

N50

A

the size of the largest contig/scaffold of which is 50% of the assembled data is in a contig/scaffold of that size or larger

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24
Q

Read length

A
  • A single read cannot span a repetitive region that is longer than the read length.
  • This prevents long contigs from forming.
  • The longer the read length the larger the repeat region that can be assembled.
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25
Q

Read depth/coverage

A
  • The average number of times each base appears in the final assembly.
  • A coverage of 10X means that each base is on average found in 10 reads.
  • The deeper the coverage, the more clearly any sequence or structure changes can be discerned from sequence error
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26
Q

Ploidy

A
  • The number of copies of the genome in the organism.
  • Bacteria =1; Human=2; Potato=4; Strawberry=8
  • The higher the ploidy, the harder it is to accurately assemble.
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27
Q

Genomic resequencing

A
  • to look for a variant
  • identify differences between strains/organisms/individuals
  • assembly against a reference is much easier than de-novo sequecing
  • may impact how you are treated medically in the future/potential of personalised medicine
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28
Q

Resequencing steps

A
  • different to reference sequence
  • gap compared to reference sequence
  • duplicated gene or region?
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29
Q

Challenges of short read re-sequencing

A
  • deleting a whole genome, hard to look for something that’s not there
  • duplication is same kind of problem
  • inversion, if sequence is short its hard to tell
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30
Q

Single molecule real time sequencing (PacBio)

A
  • long read (10kb+)
  • high error rate (14%)
  • cyclising the template means it can be read many times and an accurate consensus obtained
  • iontorrent works in a similar way but detects the pH change on nucleotide addition
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31
Q

Nanopore sequencing

A
  • as DNA is passed through the nanopore by the molecular motor under the influence of a potential
  • the current changes in a detectable way depending on the bases occluding the pore
  • the current can be interpreted to read the DNA sequence
  • to improve accuracy, a hairpin adapter is ligated to the end of the DNA fragment
  • this causes both strands of DNA to be read sequentially
32
Q

Ultra long read issues

A

Accuracy
- at present around 95-98%
Throughput
- much slower than Illumina (5Gb/48hr vs 150Gb/96hr)
Toolset
- these are new technologies and the analysis tools are still being developed

33
Q

Sequencing summary

A
  • most sequencing is by synthesis
  • current sequencing technologies can produce terabases per day
  • assembly is a challenge, especially for large genomes
  • repetitive regions are challenging
  • small changes compared to a reference are challenging
  • new technologies are helping to solve the challenge
  • careful experimental design can help solve the challenge
  • long reads - lower throughput but better for genome structure
  • short reads - higher throughput but better for sequence accuracy
34
Q

How do we differ from one another?

A

we differ from each other in small polymorphisms and structural variation

35
Q

What is a reference genome?

A

A standard sequence against which we can compare other sequences

36
Q

What are the problems with the reference?

A
  • The reference is from a very small subset of donors and is a mosaic
  • People vary, in some regions far more than others. (GRCh38 has 261 alternate scaffolds)
  • The reference is incomplete (603 gaps)
37
Q

dbSNP

A

a collection of genomic variation for human and other species

38
Q

Single nucleotide variants/polymorphisms

A
  • substitution
  • deletion
  • insertion
39
Q

Structural variants

A

changes in the overall structure of the genome

  • duplication
  • loss
  • translocation
  • inversion
  • repeat
  • deletion
40
Q

Haemophilus Influenza

A
  • a human pathogen that lives in the upper intestinal tract
  • can cause conjunctivitis and meningitis
  • sequenced in 1995
  • first whole genome sequences using a shotgun method
  • since then 28 different strains have been sequences and the pathogens have been identified
41
Q

What does the presence of genes responsible for different functions determine?

A

the virulence of the organism

42
Q

Virulence

A

the degree ofpathogenicitywithin a group or species ofparasitesas indicated by case fatality rates and/or the ability of the organism to invade the tissues of thehost

43
Q

Virulence Factors

A

genes which produce products essential for virulence

44
Q

Where do gene differences between strains tend to occur?

A
  • in clustered regions on the chromosome

- provided support for the concept of lateral gene transfer

45
Q

Lateral gene transfer

A

In some cases the genes coding for a specific cluster of genes can arise from a different source. Instead of progressive stepwise evolution, a cluster of genes from ‘foreign’ DNA is incorporated as a plasmid or integrated into the genome

46
Q

Mycoplasma genitalium

A
  • smallest known free living organism- commensal in the genitourinary tract
47
Q

Chlamydia trachomatis

A
  • human pathogen
  • causes trachoma (blindness), pharyngitis, bronchitis
  • obligate intracellular parasite transmitted by sexual contact
48
Q

How does chlamydia trachomatis effect HeLa cells by infection?

A
  • prevents fusion of phagosome and lysosome

- takes in ATP from host cells as it cannot produce it itself

49
Q

Metabolism of chlamydia trachomatis

A
  • metabolic pathways are patchy
  • part of TCA missing
  • cannot synthesise ATP
  • doesn’t appear to synthesise amino acids
  • contains a well defined recombinase pathway
  • reported to recombine and reshuffle the genome quite readily
  • contains many fatty acid and phospholipid synthesis
50
Q

How do parasitic or commensal bacteria benefit?

A
  • resources of the host
  • taking things is more efficient than making them
  • their genomes have adapted and lost key metabolic processes
51
Q

DNA replication is expensive

A

there is no competitive advantage for a bacterium to keep DNA that is of no use or redundant

52
Q

What is the minimal genome?

A
  • take a small genome and make it smaller by knocking out genes
53
Q

Transposon

A
  • ‘mobile’ DNA element that codes for enzymes that allow it to relocate in the genome
  • can be many 10’s of kb long and include many genes
54
Q

Method applied to mycoplasma genitalium

A
  • reduced essential gene count from 482 to 389
  • synthesised the entire genome and transplanted into an empty cell
  • the new synthetic organism grew
55
Q

Means of horizontal (lateral) gene transfer

A
  • virus
  • ingest DNA. foreign DNA can be taken up by a variety of mechanisms Phage infection. direct introduction of DNA into cells (competent cells).
  • ingest organism. by ingesting an organism then using its DNA
  • conjugation (mating). Exchanging DNA with a related organism. Inside the cell the DNA could stay as a plasmid or integrate into the host genome via viral integrases
  • OR transposon jumps from ingested DNA to genome (mobile DNA element)
56
Q

Identifying horizontal gene transfer

A
  • using phylogeny (horizontally acquired gene cluster)
  • using sequence properties e.g. GC content
  • genes incorporated from different sources may have different baseline GC content, or different kmer usage
57
Q

Horizontal gene transfer - organismal tree

A

genes are transferred laterally between species e.g. up and down between C and D

58
Q

Horizontal gene transfer - Gene x tree

A
  • apparent close relationship of lineages inferred from sequences of x reflects the lateral transfer of this gene rather than the phylogeny of the organisms
59
Q

Horizontal gene transfer - consensus tree

A

Based on multiple genes more accurately reflects the organismal phylogeny

60
Q

Lateral gene transfer complicates phylogenetic relationships

A
  • the phylogeny of four hypothetical prokaryote species, two of which have been involved in a lateral transfer of gene x
  • a tree based only on gene x shows the phylogeny of the laterally transferred gene, rather than the organismal phylogeny
  • a consensus tree based on multiple genes is more likely to reflect the true organismal phylogeny, especially if those genes come from a stable core of genes involved in fundamental processes
61
Q

Factors that help an organism invade the host

A 
Cell attachment
- adhesins, fimbrae etc.
Capsules
- prevent attack by macrophages and digestion
Degrading enzymes
- hyaluronidase, proteases, lipases
62
Q

Factors that help an organism evade the hosts defences

A

Toxins
- endotoxins and exotoxins
Immunosuppressants
- e.g. anti-immunoglobulin proteases

63
Q

Endotoxins

A

part of the bacterial structure e.g. lipopolysaccharide

64
Q

Exotoxins

A

secreted by bacteria e.g. shiga toxin, pertussis toxin, cholera, botox

65
Q

Virulence factors

A

toxins etc and where they are coded

66
Q

B.cereus strains

A
  • they have incorporated the virulence factors into their genome
  • do not have a plasmid
67
Q

B.anthracis

A
  • two plasmids
  • pXO1 contains the toxins
  • pXO2 produces the capsule, preventing phagocytosis and is used for immunization of domesticated animals worldwide
68
Q

Sterne strain (34F2)

A

has lost the pXO2 plasmid

69
Q

Anthrax in chimpanzees

A

In 2010 a group of researchers identified a bacterium responsible for a fatal anthrax-like disease in chimpanzees which had closer sequence similarity to B.cereus and B.turingiensis than to B.anthracis but contained the pXO1 and pXO2 plasmids (and a third plasmid.)

70
Q

RNAseq

A

sequencing all the RNA molecules in a cell

71
Q

Metagenomics

A

Sequence every organism in the environment

72
Q

Microbial genomes are minimal

A

if a gene isn’t required then it tends to be lost

73
Q

Microbial genomes reflect the biology

A

the genes tell us about the life of the organism

74
Q

Microbial genomes are plastic

A

they are reshaped with additional plasmids, transposons etc. to add new functions

75
Q

Genome sequencing is opening up…

A

… new areas for study

76
Q

Large scale sequencing can…

A

identify disease loci through genome wide association studies

BS22002 (8 decks)

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