2016-02-04 whole genome data for the analysis of food ... · whole genome data for the analysis of...

Whole genome data for the analysisof food borne infections

Martin Maiden

NIHR HPRU in Gastrointestinal InfectionsNIHR HPRU in Gastrointestinal Infections

Department of Zoology, University of Oxford

Acknowledgements

Clare Barker

Julia Bennett

Carly Bliss

Holly Bratcher

James Bray

Dorothea Hill

Lisa Rebbets

Melissa Jansen vanRensburg

Keith JolleyJames Bray

Carina Brehony

Marianne Clemence

Ali Cody

Fran Colles

Kanny DialloWTF

Sarah Earle

Suzanne Ford

Odile HarrisonWT

Sofia Hauck

Keith Jolley

Jasna Kovac

Jenny MacLennan

Noel McCarthy

Maddi Pearce

Charlene Rodrigues

Samuel Sheppard

Helen Strain

Eleanor Watkins

Helen Wimalarathna

Acknowledgements

Genome sequences and clinicalmicrobiology

• Definitive:

– all biological variation ultimately derives fromnucleotide sequence changes;

– fundamental level of information;

– any part of the genome can be accessed.

• Reproducible:

– nucleotide sequences are either right or wrong

1 gagttttatc gcttccatga cgcagaagtt aacactttcg gatatttctg atgagtcgaa61 aaattatctt gataaagcag gaattactac tgcttgttta cgaattaaat cgaagtggac121 tgctggcgga aaatgagaaa attcgaccta tccttgcgca gctcgagaag ctcttacttt181 gcgacctttc gccatcaact aacgattctg tcaaaaactg acgcgttgga tgaggagaag241 tggcttaata tgcttggcac gttcgtcaag gactggttta gatatgagtc acattttgtt301 catggtagag attctcttgt tgacatttta aaagagcgtg gattactatc tgagtccgat361 gctgttcaac cactaatagg taagaaatca tgagtcaagt tactgaacaa tccgtacgtt421 tccagaccgc tttggcctct attaagctca ttcaggcttc tgccgttttg gatttaaccg481 aagatgattt cgattttctg acgagtaaca aagtttggat tgctactgac cgctctcgtg541 ctcgtcgctg cgttgaggct tgcgtttatg gtacgctgga ctttgtggga taccctcgct601 ttcctgctcc tgttgagttt attgctgccg tcattgctta ttatgttcat cccgtcaaca661 ttcaaacggc ctgtctcatc atggaaggcg ctgaatttac ggaaaacatt attaatggcg721 tcgagcgtcc ggttaaagcc gctgaattgt tcgcgtttac cttgcgtgta cgcgcaggaa781 acactgacgt tcttactgac gcagaagaaa acgtgcgtca aaaattacgt gcggaaggag841 tgatgtaatg tctaaaggta aaaaacgttc tggcgctcgc cctggtcgtc cgcagccgtt901 gcgaggtact aaaggcaagc gtaaaggcgc tcgtctttgg tatgtaggtg gtcaacaatt961 ttaattgcag gggcttcggc cccttacttg aggataaatt atgtctaata ttcaaactgg

1021 cgccgagcgt atgccgcatg acctttccca tcttggcttc cttgctggtc agattggtcg1081 tcttattacc atttcaacta ctccggttat cgctggcgac tccttcgaga tggacgccgt1141 tggcgctctc cgtctttctc cattgcgtcg tggccttgct attgactcta ctgtagacat1201 ttttactttt tatgtccctc atcgtcacgt ttatggtgaa cagtggatta agttcatgaa1261 ggatggtgtt aatgccactc ctctcccgac tgttaacact actggttata ttgaccatgc1321 cgcttttctt ggcacgatta accctgatac caataaaatc cctaagcatt tgtttcaggg1381 ttatttgaat atctataaca actattttaa agcgccgtgg atgcctgacc gtaccgaggc1441 taaccctaat gagcttaatc aagatgatgc tcgttatggt ttccgttgct gccatctcaa1501 aaacatttgg actgctccgc ttcctcctga gactgagctt tctcgccaaa tgacgacttc1561 taccacatct attgacatta tgggtctgca agctgcttat gctaatttgc atactgacca1621 agaacgtgat tacttcatgc agcgttacca tgatgttatt tcttcatttg gaggtaaaac1681 ctcttatgac gctgacaacc gtcctttact tgtcatgcgc tctaatctct gggcatctgg1741 ctatgatgtt gatggaactg accaaacgtc gttaggccag ttttctggtc gtgttcaaca1801 gacctataaa cattctgtgc cgcgtttctt tgttcctgag catggcacta tgtttactct1861 tgcgcttgtt cgttttccgc ctactgcgac taaagagatt cagtacctta acgctaaagg1921 tgctttgact tataccgata ttgctggcga ccctgttttg tatggcaact tgccgccgcg1981 tgaaatttct atgaaggatg ttttccgttc tggtgattcg tctaagaagt ttaagattgc2041 tgagggtcag tggtatcgtt atgcgccttc gtatgtttct cctgcttatc accttcttga2101 aggcttccca ttcattcagg aaccgccttc tggtgatttg caagaacgcg tacttattcg2161 ccaccatgat tatgaccagt gtttccagtc cgttcagttg ttgcagtgga atagtcaggt

– nucleotide sequences are either right or wrongand this can be checked;

– reverse mutations are (usually) rare.

• Scalable:

– nucleotide sequencing technology can beconducted on one or many samples and on a fewbase pairs or a whole genome.

• Manipulable:

– nucleotide sequences;

– can be analysed with model-based methods.

2221 taaatttaat gtgaccgttt atcgcaatct gccgaccact cgcgattcaa tcatgacttc2281 gtgataaaag attgagtgtg aggttataac gccgaagcgg taaaaatttt aatttttgcc2341 gctgaggggt tgaccaagcg aagcgcggta ggttttctgc ttaggagttt aatcatgttt2401 cagactttta tttctcgcca taattcaaac tttttttctg ataagctggt tctcacttct2461 gttactccag cttcttcggc acctgtttta cagacaccta aagctacatc gtcaacgtta2521 tattttgata gtttgacggt taatgctggt aatggtggtt ttcttcattg cattcagatg2581 gatacatctg tcaacgccgc taatcaggtt gtttctgttg gtgctgatat tgcttttgat2641 gccgacccta aattttttgc ctgtttggtt cgctttgagt cttcttcggt tccgactacc2701 ctcccgactg cctatgatgt ttatcctttg aatggtcgcc atgatggtgg ttattatacc2761 gtcaaggact gtgtgactat tgacgtcctt ccccgtacgc cgggcaataa cgtttatgtt2821 ggtttcatgg tttggtctaa ctttaccgct actaaatgcc gcggattggt ttcgctgaat2881 caggttatta aagagattat ttgtctccag ccacttaagt gaggtgattt atgtttggtg2941 ctattgctgg cggtattgct tctgctcttg ctggtggcgc catgtctaaa ttgtttggag3001 gcggtcaaaa agccgcctcc ggtggcattc aaggtgatgt gcttgctacc gataacaata3061 ctgtaggcat gggtgatgct ggtattaaat ctgccattca aggctctaat gttcctaacc3121 ctgatgaggc cgcccctagt tttgtttctg gtgctatggc taaagctggt aaaggacttc3181 ttgaaggtac gttgcaggct ggcacttctg ccgtttctga taagttgctt gatttggttg3241 gacttggtgg caagtctgcc gctgataaag gaaaggatac tcgtgattat cttgctgctg3301 catttcctga gcttaatgct tgggagcgtg ctggtgctga tgcttcctct gctggtatgg3361 ttgacgccgg atttgagaat caaaaagagc ttactaaaat gcaactggac aatcagaaag3421 agattgccga gatgcaaaat gagactcaaa aagagattgc tggcattcag tcggcgactt3481 cacgccagaa tacgaaagac caggtatatg cacaaaatga gatgcttgct tatcaacaga3541 aggagtctac tgctcgcgtt gcgtctatta tggaaaacac caatctttcc aagcaacagc3601 aggtttccga gattatgcgc caaatgctta ctcaagctca aacggctggt cagtatttta3661 ccaatgacca aatcaaagaa atgactcgca aggttagtgc tgaggttgac ttagttcatc3721 agcaaacgca gaatcagcgg tatggctctt ctcatattgg cgctactgca aaggatattt3781 ctaatgtcgt cactgatgct gcttctggtg tggttgatat ttttcatggt attgataaag3841 ctgttgccga tacttggaac aatttctgga aagacggtaa agctgatggt attggctcta3901 atttgtctag gaaataaccg tcaggattga caccctccca attgtatgtt ttcatgcctc3961 caaatcttgg aggctttttt atggttcgtt cttattaccc ttctgaatgt cacgctgatt4021 attttgactt tgagcgtatc gaggctctta aacctgctat tgaggcttgt ggcatttcta4081 ctctttctca atccccaatg cttggcttcc ataagcagat ggataaccgc atcaagctct4141 tggaagagat tctgtctttt cgtatgcagg gcgttgagtt cgataatggt gatatgtatg4201 ttgacggcca taaggctgct tctgacgttc gtgatgagtt tgtatctgtt actgagaagt4261 taatggatga attggcacaa tgctacaatg tgctccccca acttgatatt aataacacta4321 tagaccaccg ccccgaaggg gacgaaaaat ggtttttaga gaacgagaag acggttacgc4381 agttttgccg caagctggct gctgaacgcc ctcttaagga tattcgcgat gagtataatt4441 accccaaaaa gaaaggtatt aaggatgagt gttcaagatt gctggaggcc tccactatga4501 aatcgcgtag aggctttgct attcagcgtt tgatgaatgc aatgcgacag gctcatgctg4561 atggttggtt tatcgttttt gacactctca cgttggctga cgaccgatta gaggcgtttt4621 atgataatcc caatgctttg cgtgactatt ttcgtgatat tggtcgtatg gttcttgctg4681 ccgagggtcg caaggctaat gattcacacg ccgactgcta tcagtatttt tgtgtgcctg4741 agtatggtac agctaatggc cgtcttcatt tccatgcggt gcactttatg cggacacttc4801 ctacaggtag cgttgaccct aattttggtc gtcgggtacg caatcgccgc cagttaaata4861 gcttgcaaaa tacgtggcct tatggttaca gtatgcccat cgcagttcgc tacacgcagg4921 acgctttttc acgttctggt tggttgtggc ctgttgatgc taaaggtgag ccgcttaaag4981 ctaccagtta tatggctgtt ggtttctatg tggctaaata cgttaacaaa aagtcagata5041 tggaccttgc tgctaaaggt ctaggagcta aagaatggaa caactcacta aaaaccaagc5101 tgtcgctact tcccaagaag ctgttcagaa tcagaatgag ccgcaacttc gggatgaaaa5161 tgctcacaat gacaaatctg tccacggagt gcttaatcca acttaccaag ctgggttacg5221 acgcgacgcc gttcaaccag atattgaagc agaacgcaaa aagagagatg agattgaggc5281 tgggaaaagt tactgtagcc gacgttttgg cggcgcaacc tgtgacgaca aatctgctca5341 aatttatgcg cgcttcgata aaaatgattg gcgtatccaa cctgca

Sanger, F., Air, G. M., Barrell, B. G., Brown, N. L.,Coulson, A. R., Fiddes, C. A., Hutchison, C. A.,Slocombe, P. M. & Smith, M. (1977). Nucleotidesequence of bacteriophage phi X174 DNA. Nature265, 687-695.

Frederick Sanger (1918-2013)

Questions in clinical microbiology

Centuries+ decades years months weeks days hours

evolution emergence epidemiology diagnosis

Relative discrimination required HighLow

Relative amount of genetic change LowHigh

Mice andmen

Vibrio cholerae

Yersinia pestis

Escherichia coli

Plants

Bacterial genome diversityGene pool

Accessory

Accessory genome:e.g. alternativemetabolic pathways,transport systems.

Core genome:e.g. DNA replication,ribosomes, cellenvelope, keymetabolic pathways.

Mycoplamagenitalium

Streptococcuspneumoniae

Bacillusanthracis

Mycobacteriumtuberculosis

Campylobacterjejuni

Neisseriameningitidis

TreponemapallidumChlamydia

pneumoniae

Rickettsiaprowazekii

Staphylococcusaureus

Genetic elements may be subject to stabilising (negative) or diversifying(positive) selection or be neutral (rare in most bacteria).

Parasitic elements(phages plasmids)e.g. toxins,restriction/modificationsystems.

Core

Mobileelements

Gene pool:e.g. antibioticresistance, degrativemetabolism.

Patterns in sequence variationa primer on bacterial population biology

Ideas about bacterialpopulations are dominated thefacts that bacteria:• are asexual;• reproduce by binary fission,

with each ‘mother’ cellwith each ‘mother’ cellgiving rise to two identical‘daughter’ cells (clones).

• accumulate genetic changeby ‘vertical’ inheritance. Gupta, S. & Maiden, M.C.J. Exploring

the evolution of diversity in pathogenpopulations. Trends in Microbiology 9,181-192 (2001).

The clonal population model:asexuality with diversity reduction

Diversity reduction

periodicselection

Original genotype

Mutant genotype

selection

bottlenecking

Levin BR. 1981. Periodicselection, infectious geneexchange and the geneticstructure of E. coli populations.Genetics 99(1):1-23.

Bacterial populations should, therefore, be easy to understand …

Molecular typing made easy – theclonal frame

Years

100s+

decades years weeks/

months

hours/

days

AAAA

ATAT

ATAT

Progressive accumulation of genetic change

AAAA

ATAA

ATAT

ATAA

TTAT

TTTT

Impact of recombination on bacterialpopulation structure

Horizontal genetic

Original genotype

Recombinantgenotype

Recombination disrupts clonal structure, disrupting tree-likephylogeny, linkage disequilibrium and congruence.

Maynard Smith J, Dowson CG, Spratt BG. 1991. Localized sex inbacteria. Nature 349:29-31.

Horizontal geneticTransfer ('localisedsex')

Clonal and non-clonal populationstructures

Clonal

• Linkage disequilibrium

– non-random allelecombinations.

• Tree-like phylogeny

Non-clonal

• Linkage equilibrium

– random allelecombinations.

• Net-like phylogeny• Tree-like phylogeny

– a bifurcating tree accuratelymodels descent.

• Congruence

– the same phylogentic signalis recorded throughout thegenome

• Net-like phylogeny

– a bifurcating treecannot model descent.

• Incongruence

– different phylogenticsignals are recordedthroughout the genome

A spectrum of population structuresStrictly clonal Fully non-clonal

In practice, different levels of clonal signal is observed in

C. jejuni H. pyloriS. entericaS. Typhi

In practice, different levels of clonal signal is observed indifferent bacterial populations.It is thought that this is a consequence of differingrelative rates of recombination to mutation, althoughother forces may play a role.

Gupta, S. & Maiden, M.C.J. Exploring the evolution ofdiversity in pathogen populations. Trends in Microbiology9, 181-192 (2001).

Dealing with recombination

• Recombination violates the assumptions of clonalevolution.

• This has to be accounted for in models ofbacterial evolution by:– Ignoring it (works if recombination is very low);– Ignoring it (works if recombination is very low);– Identifying (always an estimation) and

• removing possible recombination (e.g. GUBBINS), or• Including recombination and mutation in calculation of

phylogenies (CLONALFRAME and CLONALFRAMEML);

– Using alleles as the unit of analysis (gene-by-geneanalysis).

Campylobacter sequence typing

tkt

gltA

aspA

uncA

pgm

10 20 30 40 50aspA1 ATGATAGGTGAAGATATACAAAGAGTATTAGAAGCTAGAAAATTGATTTTaspA2 ..................................................aspA3 ..................................................aspA4 ........C...................................A.....aspA6 ..................................................aspA7 ..................................................aspA8 ..................................................aspA9 ..................................................aspA10 ........C...................................A.....aspA14 ..................................................aspA16 ..................................................aspA17 ..................................................

• Campylobacter Seven-locus STsummarises 3,309bp of data asan allelic profile: e.g. ST-45: 4-7-10-4-1-7-1

• This is 0.2% of the genome.

Dingle, K. E., Colles, F. M., Wareing, D. R. A., Ure, R., Fox, A. J., Bolton, F. J., Bootsma,H. J., Willems, R. J. L., Urwin, R. & Maiden, M. C. J. (2001). Multilocus sequence typingsystem for Campylobacter jejuni. J Clin Microbiol 39, 14-23.

glyA

gln

A

porA

fla

aspA18 ..................................................aspA19 ..................................................aspA20 ........C.........................................aspA21 ..................................................aspA22 ..................................................aspA23 ..................................................aspA24 ..................................................aspA26 ..................................................aspA27 ..................................................aspA28 .................G................................aspA30 ............................................A.....aspA31 ..................................................aspA32 .....G....................T...............C.......aspA33 .....G....................T...............C.......aspA34 ..................................................aspA48 ........C...................................A.....aspA64 ..................................................

• 7,763 STs from 33,051 isolatesin PubMLST database (May2015).

• 400-744 alleles per locus.

• Many polymorphisms per locus.

Polymorphisms in the uncA MLST locus

Relationships among STs:clonal complexes

ST-45, 26 isolates

ST-4917, 1 isolate4-280-10-4-1-7-1

ST-1701, 1 isolate4-7-10-4-1-51-1

glnA

tkt

3nt

1 ntST-45, 26 isolates4-7-10-4-1-7-1

4-7-10-4-1-51-1

ST-137, 9 isolates4-7-10-4-42-7-1

ST-583, 4 isolates4-7-10-4-42-51-1

ST-2219, 2 isolates10-7-10-4-1-7-1

ST-4852, 1 isolate37-7-10-4-1-7-1

ST-5086, 1 isolate7-7-10-4-1-7-1

tkt

tkt

pgm pgm

aspA

Colles, F. M. & Maiden, M. C. J. (2012). Campylobacter sequence typing:applications and future prospects. Microbiology. 158(11): 2695-2709.

2 nt

7 nt

9 nt

1 nt

60

70

80

90

100

Pe

rce

nt

of

Iso

late

s

humans

poultry

cattle

sheep

pigs

starlings

sand

Dingle, K. E., Colles, F.M., Ure, R., Wagenaar,J., Duim, B., Bolton, F.J., Fox, A. J., Wareing,D. R. A. & Maiden, M.C. J. (2002). Molecularcharacterisation ofCampylobacter jejuni

Campylobacter clonal complexes:association with isolation source

0

10

20

30

40

50

ST-45 ST-61 ST-403 ST-177 ST-179

Clonal Complex

Pe

rce

nt

of

Iso

late

s

sand

Number of isolates: 814; 17 clonal complexes in total, 5 shown.

Campylobacter jejuniclones: a rational basisfor epidemiologicalinvestigations. EmergInfect Dis 8, 949-955.

15

20

25

30

35

40

Pro

po

rtio

nCampylobacter in bird species

0

5

10

85

5

19

10

89

95

8

81

3

83

0

13

57

12

29

13

24

12

90

12

52

13

37

63

7

12

25

13

22

12

61

13

01

13

35

53

8

12

76

12

94

99

3

12

57

12

99

99

4

13

23

12

93

13

20

13

52

13

55

13

28

13

09

12

95

68

2

12

84

ST

Chicken Blackbird Gull Owl Starling Thrush

Mallard Dunlin Redwing Fieldfare Sandpiper Jackdaw

Stint Wagtail Reed warbler Sparrowhawk Woodcock Yellowhammer

Griekspoor, P., Colles, F. M., Mccarthy, N. D., Hansbro, P. M., Ashhurst-Smith, C., Olsen, B.,Hasselquist, D., Maiden, M. C. J. & Waldenstrom, J. (2013). Marked host specificity and lack ofphylogeographic population structure of Campylobacter jejuni in wild birds. Mol Ecol 22, 1463-1472.

Campylobacter Genealogies and clonalcomplexes

21 28345

22

48

353

682

443

354

ST-21 Complex

ST-257Complex

ST-45Complex

ST-42Complex

0.02

0.02

177

403

179

52

42

C. doylei

1275206

433

257

61

443

ST-61Complex

Bovine/ovine

Environment

Human disease

Chicken

Both

Sheppard, S. K., Colles, F. M., McCarthy, N. D., Strachan, N. J., Ogden, I. D., Forbes, K.J., Dallas, J. F. & Maiden, M. C. (2011). Niche segregation and genetic structure ofCampylobacter jejuni populations from wild and agricultural host species. Mol Ecol 20,3484-3490.

Disease attribution

Potential sources ofHuman infection.

Probabilistic assignment ofisolates to hosts.

Different hosts have different pools ofCampylobacter MLST alleles, so diseasecan be attributed at each of the seven loci.

Clinical isolategenotypeprobably camefrom cattle.

Quality of assignment depends principally onthe quality of the reference dataset; there area variety of statistical models available.

Attribution of Campylobacter tochicken, UK & NZ

Wilson, D. J., Gabriel, E., Leatherbarrow, A. J. H., Cheesbrough, J., Gee, S., Bolton, E., Fox, A., Fearnhead, P., Hart, A. & Diggle, P. J. (2008).Tracing the source of campylobacteriosis. PLoS Genet 26, e1000203.Sheppard, S. K., Dallas, J. F., Strachan, N. J., MacRae, M., McCarthy, N. D., Wilson, D. J., Gormley, F. J., Falush, D., Ogden, I. D., Maiden, M.C. & Forbes, K. J. (2009). Campylobacter genotyping to determine the source of human infection. Clin Infect Dis 48, 1072-1078.Mullner, P., Spencer, S. E., Wilson, D. J., Jones, G., Noble, A. D., Midwinter, A. C., Collins-Emerson, J. M., Carter, P., Hathaway, S. & French,N. P. (2009). Assigning the source of human campylobacteriosis in New Zealand: A comparative genetic and epidemiological approach. InfectGenet Evol. 138:1372-83.

Data sources

First generation ‘Next generation’

Archival Short-read sequence

data

Bacterialisolate

Clinicalspecimen

Contiguous sequences (contigs.)

DNA

Sequence onpreferred platform

(e.g. Illumina)

Complete, assembled closed genomeswith annotation, available from public

databases (e.g. IMGD)

TGGAGCAGATCGAGGAGAGCGAGTTCGACGC







TGGAGCAGATCGAGGAGAGCGAGTTCGACGCTGGAGCAGATCGAGGAGAGCGAGTTCGACGC

Assemble withpreferred software

(e.g. VELVET)

Approaches to the analysis of wholegenome sequence data

• Comparison of short reads to a reference– ‘SNP’ calling: compares short reads to a high quality reference,

particularly used in comparing very closely related isolates.

• De novo assembly and comparison– ‘k-mer’ approach: reference-free assembly and comparison,– ‘k-mer’ approach: reference-free assembly and comparison,

independent of biological information.

• Gene-by-gene analysis– ‘whole genome MLST (wgMLST)’: de novo reference free or guided

assembly, followed by locus by locus identification and comparison ofgenetic variation.

Maiden, M. C., van Rensburg, M. J., Bray, J. E., Earle, S. G., Ford, S. A., Jolley,K. A. & McCarthy, N. D. (2013). MLST revisited: the gene-by-gene approach tobacterial genomics. Nat Rev Microbiol. 11(10): 728-36.

Whole-genome sequencing (WGS) at thepopulation scale

Bacterialspecimens

Tagged DNA

Illumina pairedend sequencingend sequencing

de novo Velvetassembly

Deposition intoBIGSDB

Annotation byautotagging at >1600loci, web publication

Sheppard, S. K., Jolley, K. A. & Maiden, M. C. J.(2012). A Gene-By-Gene Approach to BacterialPopulation Genomics: Whole Genome MLST ofCampylobacter. Genes 3, 261-277.

Rapid automated genome assembly

506 IsolatesIllumina Genome Analyzer GAIIxRead Lengths: 100 NucleotidesAverage Input FASTQ Filesize:586MB

(258 million nucleotides)

Pro

gram

Tim

e(h

h:m

m:s

s) Total AutoAssembler.pl Program TimeUsing 10 Threads Per Assembly

(258 million nucleotides)Average Number of Reads: 2.58millionK-mer Range: 21-99

Median Final K-mer: 81Median N50: 37,503Average Number of Contigs: 209Average Program Time: 22 mins 31secsTotal Program Time: 58 hours

Filesize (MB)

Pro

gram

Tim

e(

Bray, J., Jolley, K.A. Maiden, M.C.,unpublished

Population genomics:the gene-by-gene approach

CompleteSequence

Contigs

Annotation

Bacterial IsolateGenome SequenceDatabase (BIGSDB)

Gene sequencesProvenance/phenotypeinformation

Bratcher, H. B., Bennett, J. S. & Maiden, M. C. J. (2012). Evolutionary and genomic insights intomeningococcal biology. Future Microbiol 7, 873-885.Sheppard, S. K., Jolley, K. A. & Maiden, M. C. J. (2012). Whole Genome MLST of Campylobacter: aGene-by-gene approach. Genes 3, 261-277.

Bacterial Isolate Genome Sequence Database(BIGSDB)

CCATCCCGTTGTCGAACAGCAGGTACGCCA

CTTCACCGCCAACCACACCGACCTTGACCAC

AAACACCGCCTCATGCTGCTCACCGGCCCC

AATATGGGCGGCAAATCCACCTACATGCGCAGGAACCCTCAAAGCCGTTTTCCCGGAAAACCTATCCACAGCCGAACAGCTCCGCCAAGCCA

TTTTGCCCGAACCTTCCGTCTGGCTGAAAGACGGCAATGTCATCAACCACGGTTTTCATCCC

GAACTGGACGAATTGCGCCGCATTCAAAACCATGGCGACGAATTTTTGCTGGATTTGGAAGCCAAGGAACGCGAACGTACCGGTTTGTCCAC

ACTTAAAGTCGAGTTCAACCGCGTTCACGGCTTTTACATTGAATTGTCCAAAACCCAAGCCG

AACAAGCACCTGCCGACTACCAACGCCGGCAAACCCTTAAAAACGCCGAACGCTTCATCACGCCGGAACTGAAAGCCTTTGAAGACAAAGT

GCTGACTGCTCAAGAGCAAGCCCTCGCCTTAGAAAAACAACTCTTTGACGGCGTATTGAAA

AACCTTCAGACGGCATTGCCGCAGCTTCAAAAAGCCGCCAAAGCCGCCGCCGCGCTGGACGTGTTGTCCACATTTTCAGCCTTGGCAAAAG

AGCGGAACTTCGTCCGCCCCGAGTTTGCCG

ACAAGTCGCGCTGATTGTTT

AACCTTCAGACGGCATTGCCGCAGCTTCAAA

AAGCCGCCAAAGCCGCCGCCGCGCTGGACGTGTTGTCCACATTTTCAGCCTTGGCAAAAG

AGCGGAACTTCGTCCGCCCCGAGTTTGCCGACTATCCGGTTATCCACATCGAAAACGGCCGCCATCCCGTTGTCGAACAGCAGGTACGCCA

CTTCACCGCCAACCACACCGACCTTGACCACAGGAACCCTCAAAGCCGTTTTCCCGGAAAAC

CTATCCACAGCCGAACAGCTCCGCCAAGCCATTTTGCCCGAACCTTCCGTCTGGCTGAAAGACGGCAATGTCATCAACCACGGTTTTCATCC

CGAACTGGACGAATTGCGCCGCATTCAAAACCATGGCGACGAATTTTTGCTGGATTTGGAAG

CCAAGGAACGCGAACGTACCGGTTTGTCCACACTTAAAGTCGAGTTCAACCGCGTTCACGGCTTTTACATTGAATTGTCCAAAACCCAAGCC

GCCCCGAGTTTGCCGACTATCCGGTTATCCACATCGAAAACGGCCGCCATCCCGTTGTCGA

ACAGCAGGTACGCCACTTCACCGCCAACCACACCGACCTTGACCACAAACACCGCCTCATGCTGCTCACCGGCCCCAATATGGGCGGCAAA

TCCACCTACATGCGCCAAGTCGCGCTGATTGTTT

AGGAACCCTCAAAGCCGTTTTCCCGGAAAAC

CTATCCACAGCCGAACAGCTCCGCCAAGCCATTTTGCCCGAACCTTCCGTCTGGCTGAAAGACGGCAATGTCATCAACCACGGTTTTCATCC

CGAACTGGACGAATTGCGCCGCATTCAAAACCATGGCGACGAATTTTTGCTGGATTTGGAAG

CCAAGGAACGCGAACGTACCGGTTTGTCCACACTTAAAGTCGAGTTCAACCGCGTTCACGGCTTTTACATTGAATTGTCCAAAACCCAAGCC

GAACAAGCACCTGCCGACTACCAACGCCGGCAAACCCTTAAAAACGCCGAACGCTTCATCA

CGCCGGAACTGAAAGCCTTTGAAGACAAAGTGCTGACTGCTCAAGAGCAAGCCCTCGCCTTAGAAAAACAACTCTTTGACGGCGTATTGAAA

AACCTTCAGACGGCATTGCCGCAGCTTCAAAAAGCCGCCAAAGCCGCCGCCGCGCTGGAC

GTGTTGTCCACATTTTCAGCCTTGGCAAAAGAGCGGAACTTCGTCCGCCCCGAGTTTGCCGACTATCCGGTTATCCACATCGAAAACGGCCG

CCATCCCGTTGTCGAACAGCAGGTACGCCACTTCACCGCCAACCACACCGACCTTGACCAC

AAACACCGCCTCATGCTGCTCACCGGCCCCAATATGGGCGGCAAATCCACCTACATGCGCCAAGTCGCGCTGATTGTTT

abcZadkaroEfumCgdhpdhCpgmporAporB

Sequencebin

Locusdefinitions

tables: porBfetApenArpoB16SLocus XLocus Y

Jolley, K. A. & Maiden, M. C. (2010). BIGSdb: Scalableanalysis of bacterial genome variation at thepopulation level. BMC Bioinformatics 11, 595.

tables:annotation

source Locus Allele Provenance

abcZ 2 Country UK

adk 3 Year 2013

aroE 4 serogroup B

gdh 8 Disease carrier

pdhC 4 Age 23

pgm 6 Source Swab

... etc... ... etc ...

Genome annotation

• Genome-wide MLST

• Autotagger – runsregularly – tags all lociwith known alleles

• Each unique sequence• Each unique sequencegiven new allelenumber

• Loci grouped in toschemes

Sheppard, S. K., Jolley, K. A. & Maiden, M. C. J. (2012). A Gene-By-Gene Approach to Bacterial Population Genomics: Whole Genome MLSTof Campylobacter. Genes 3, 261-277.

GENOMECOMPARATOR: rapid comparativegenomics

Jolley, K. A., Hill, D. M., Bratcher, H. B., Harrison, O. B., Feavers, I. M., Parkhill, J. & Maiden, M. C.(2012). Resolution of a meningococcal disease outbreak from whole genome sequence data with rapidweb-based analysis methods. J Clin Microbiol. 50(9):3046-53.

SPLITSTREE 4.0NEIGHBORNET

Ribosomal multi-locus sequencetyping, rMLST

• Isolate characterisation from ‘domain to strainfrom WGS data.

• Indexes the 53 universally presentribosomal genes.

• September 2015 ribosomal alleles defined for:• >129,791 genome sequences;

Jolley, K. A., Bliss, C. M., Bennett, J. S., Bratcher, H. B., Brehony, C. M., Colles, F. M.,Wimalarathna, H. M., Harrison, O. B., Sheppard, S. K., Cody, A. J. & Maiden, M. C. (2012).Ribosomal Multi-Locus Sequence Typing: universal characterisation of bacteria fromdomain to strain. Microbiology 158, 1005-1015.

• >129,791 genome sequences;• 1,169 genera;• 3,434 unique species ;

• rSTs defined for 25 taxa;• Neisseria and Campylobacter to clonal

complex and Salmonella to the Serovarlevel.

MLST(7 loci)

16S rRNAsequences

(1 locus)

Ribosomal MLST(53 loci)

Family

Order

Class

Phylum

Genus

Whole genomeMLST

(>500 loci)- Core genome MLST- Accessory genome

MLST

Sequence data and nomenclature

(7 loci)

Strain

Lineage/Clonal Complex

Species

Clone

Meroclone

Maiden M.C.J. et al. 2013.MLST revisited: the gene-by-gene approach to bacterialgenomics. Nat RevMicrobiol. 2013 Sep 2. doi:10.1038/nrmicro3093.

Data submitters:currently >1300;

Data curators:currently >90 MLST schemes

Sequence definitionsMLST, rMLST, antigengenes, core genome,pan-genome

Isolate datasets

• provenance• phenotype• gene content• allelic variation• genomes

Populationannotation

• locus classification• description• biochemical

pathway

Comparativegenomics

PubMLST1998*, 2003

Gene-by-gene analysisusing reference genomeor defined loci

Gen

eA

Gen

eB

Gen

eC

Gen

eD

Allele1: TTTGATACTGTTGCCGAAGGTTTAllele2: TTTGATACCGTTGCCGAAGGTTTAllele3: TTTGATTCCGTTGCCGAAGGTTT

>750 citations

• genomes

Linked to:

pathway• Core + accessory

genome analysis• Association studies

Molecular typingSpecies identification

EpidemiologyVaccine coverage/impact

Linking genotypeto phenotype

Outbreak investigationPopulation structure

>8000 unique visitors/month*http://mlst.zoo.ox.ac.uk

Allele or nucleotide-based analysis?Campylobacter species in Cape Town

C. jejuni

Allele basedrMLST

C. jejuni subsp. doylei

C. coli

C. lariC. hominisC. fetus

C. concisusC. curvus

C. upsaliensis

Melissa Jansen van Rensburg, Unpublished.

rMLST

Campylobacter species in Cape Town

C. jejuni

C. jejuni subsp. doylei

C. coli

C. lari

Nucleotide-basedrMLST analysis

C. hominis

C. fetus

C. concisus

C. curvus

C. upsaliensis

Melissa Jansen van Rensburg, Unpublished.

Campylobacteriosis in Oxfordshire

• John Radcliffe Hospital Microbiologylaboratory: 800-900 Campylobacterisolates per year.

• Catchment area contiguous,Population ~600,000, about 1% of UKtotal.total.

• Ongoing surveillance since 2003:7,101 isolates (June 2015).

• Routine genomic surveillance ofCampylobacter isolates since June2011, 3,562 WGS (June 2015).

Cody, A. J., McCarthy, N. M., Wimalarathna, H. L., Colles, F. M., Clark, L., Bowler, I. C., Maiden, M. C. & Dingle, K. E. (2012). Alongitudinal six-year study of the molecular epidemiology of clinical Campylobacter isolates in Oxfordshire, UK. J Clin Microbiol 50,3193-3201.Cody, A. J., McCarthy, N. D., Jansen van Rensburg, M., Isinkaye, T., Bentley, S., Parkhill, J., Dingle, K. E., Bowler, I. C., Jolley, K. A. &Maiden, M. C. (2013). Real-time genomic epidemiology of human Campylobacter isolates using whole genome multilocus sequencetyping. J Clin Microbiol 51, 2526-2534.

25

30

35

Pe

rce

nta

geo

fis

ola

tes

2003-2004 2004-2005 2005-2006 2006-2007 2007-2008 2008-2009 2009-2010 2010-2011 2011-2012 2012-2013

Oxfordshire human campylobacteriosisisolates 2003-2013

0

5

10

15

20

ST-21

ST-25

7

ST-44

3

ST-45

ST-35

3

ST-48

ST-57

4

ST-20

6

ST-35

4

ST-65

8

ST-61

ST-42

ST-22

ST-60

7

ST-57

3

ST-49

ST-28

3

ST-46

4

ST-40

3

ST-52

ST-66

1

ST-10

34

ST-50

8

UA

Pe

rce

nta

geo

fis

ola

tes

Clonal complex

STRUCTURE attribution of clinicalisolates from Oxfordshire 2003-2013

Retail chicken, yellow; cattle, dark blue; sheep, light blue; wild bird sources, brown.

Cody, A. J., McCarthy, N. D., Bray, J. E., Wimalarathna, H. M., Colles, F. M., van Rensburg, M. J., Dingle, K. E., & Maiden, M.C. Unpublished

Oxfordshire isolates attributed to wildbird sources (brown)

98%

99%

100%

Perc

en

tag

eo

fis

ola

tes

94%

95%

96%

97%

2003-2004(n=532)

2004-2005(n=476)

2005-2006(n=493)

2006-2007(n=542)

2007-2008(n=430)

2008-2009(n=543)

2009-2010(n=450)

2010-2011(n=713)

2011-2012(n=786)

2012-2013(n=663)

Perc

en

tag

eo

fis

ola

tes

Year of study

Cody, A. J., McCarthy, N. D., Bray, J. E., Wimalarathna, H. M., Colles, F. M., van Rensburg, M. J., Dingle, K. E., Waldenstrom,J. & Maiden, M. C. (2015). Wild bird associated Campylobacter jejuni isolates are a consistent source of human disease, inOxfordshire, United Kingdom. Environ Microbiol Rep. 2015 Jun 24. doi: 10.1111/1758-2229.12314. [Epub ahead of print]

Seasonality by wild bird family

Oxfordshire 2003-13 Hants and Notts (2000-03)

Anatidae (mallards, geese), brown; Laridae (gulls), green; Turdidae (blackbirds, songthrush),purple; Sturnidae (starlings), pink; Scolopacidae (dunlin, sharp-tailed sandpipers), turquoise.

cgMLST 71,631 pair-wise comparisons of379 isolates at 1026 loci

8000

10000

12000P

air

wis

eco

mp

ari

so

ns

0

2000

4000

6000

25

50

75

100

125

150

175

200

225

250

275

300

325

350

375

400

425

450

475

500

525

550

575

600

625

650

675

700

725

750

775

800

825

850

875

900

925

950

975

1000

1026

Pair

wis

eco

mp

ari

so

ns

Number of 1026 loci compared which had different alleles

Cody, A. J., McCarthy, N.D., Jansen van Rensburg, M., Isinkaye, T., Bentley, S., Parkhill, J., Dingle, K. E,Bowler, I. C. W., Jolley, K.A. & Maiden, M. C. J. Clin. Microbiol., In the press.

379 isolates, 3 months Oxfordshire

Cody, A. J., McCarthy, N. D., Jansen van Rensburg, M., Isinkaye, T., Bentley, S., Parkhill, J., Dingle, K. E., Bowler, I. C.,Jolley, K. A. & Maiden, M. C. (2013). Real-time genomic epidemiology of human Campylobacter isolates using wholegenome multilocus sequence typing. J Clin Microbiol 51, 2526-2534.

Global diversity of Campylobacter isolates,humans and animals over 10 years

ST-828 CC

ST-508 CC

ST-45 CC

ST-61 CCST-22 CC

ST-42 CCST-403 CC

ST-52 CC

ST-658 CC

ST-353 CC

ST-677 CCST-179 CC

ST-1275 CC

Lefebure, T., Pavinski Bitar, P. D., Suzuki, H. & Stanhope, M. J. (2010). EvolutionaryDynamics of Complete Campylobacter Pan-Genomes and the Bacterial Species Concept.Genome Biology and Evolution 2, 646-655.

ST-206 CC

ST-48 CC

ST-21 CC

ST-443 CCST-446 CC

ST-1150 CC

NEIGHBORNET allele-based wgMLST:‘Strain 3’ isolates


NEIGHBORNET allele-based wgMLST:‘Strain 3A’ isolates


Outbreak: single diverse source

Abid, M., Wimalarathna, H., Mills, J., Saldana,L., Pang, W., Richardson, J. F., Maiden, M. C. &McCarthy, N. D. (2013). Duck Liver-associatedOutbreak of Campylobacteriosis amongHumans, United Kingdom, 2011. Emerg InfectDis 19, 1310-1313.

Outbreak: single uniform source

Fernandes, A. M., Balasegaram, S., Willis, C., Wimalarathna, H. M., Maiden, M. C. & McCarthy, N. D.(2015). Partial Failure of Milk Pasteurization as a Risk for the Transmission of Campylobacter From Cattleto Humans. Clin Infect Dis. Advance Access published June 30, 2015

Genomics in the courts...

Hierarchical gene-by-gene tracking ofCampylobacter

Centuries+ decades years months weeks days hours

Evolution emergence epidemiology diagnosis

OXC6289

OXC6393

OXC6459

Cluster 3

Relative discrimination required HighLow

Relative amount of genetic change LowHigh

OXC6565

OXC6527

OXC6530

OXC6543

OXC6524

OXC6636

OXC6590

OXC6461

OXC6393

OXC6598

OXC6600

Cluster 1

Cluster 2

Cluster 3

2016-02-04 whole genome data for the analysis of food ... · whole genome data for the analysis of...

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