Whole genome sequencing (WGS) - there s a new tool in town. Henrik Hasman DTU - Food

Transcription

1 Whole genome sequencing (WGS) - there s a new tool in town Henrik Hasman DTU - Food

2 Welcome to the NGS world TODAY Welcome Introduction to Next Generation Sequencing DNA purification (Hands-on) Lunch (Sandwishes Hands-on) DNA quantification for NGS (Hands-on) Coffee.and.Library preparation ( at the movies ) Running the MiSeq (Show n tell) Computer exercises (Hands-on) Goodbye

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4 EU RL workshop on WGS Advanced NGS on antimicrobial resistant bacteria One day the training course will consist of hands-on and theoretical teaching focusing at NGS. This training aims at introducing relevant tools for the attendees to prepare for the challenges of genomic techniques. This theme will teach you how to prepare genomic DNA from bacterial culture for DNA sequencing, give a theoretical introduction to NGS sequencing including library preparation and running the MiSeq DNA sequencer. Finally, you will also perform an exercise regarding analysis of NGS data in relation to species identification, antimicrobial resistance gene detection and plasmid typing.

5 DNA sequencing 5

6 DNA sequencing Applied Biosystems (ABI) Genetic analyser First Generation Sequencing machine (capillary Sanger sequencing) 6

7 7

8 Second generation sequencing

9 Second generation sequencing machines 454 Life Sciences (Roche) First Next Generation Sequencing machine Illumina HiSeq/GAII systems High throughput systems Ion Torrent PGM system Low/medium throughput system Illumina MiSeq system Medium throughput system 9

10 Workflow today at the clinical laboratory

11 Workflow with WGS at the clinical laboratory Didelot et al, 2012.

12 Server side Client side Raw DNA sequences Rough assembly and compression Fine assembly Identification Gene finding Comparison Summary of: What it is? Has it been seen before? How we can fight/treat? What is new/unusual? What is already known? Pathogenicity islands Virulence genes Resistance genes MLST type What is novel? Vaccine targets Virulence genes Resistance genes SNPs Google maps like view Reports Outbreaks

13 Illumina MiSeq system Medium throughput system

14 MiSeq Workflow Gram+ Analysis tools Library DNA purification DNA barcoding

15 Tutorial on MiSeq workflow MiSeq Sequencing Chemistry: ca. 20 min

16 DNA purification EasyDNA from Invitrogen

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18 Qubit DNA quantification

19 Questions? Then to the dungeons

20 MiSeq Workflow Gram+ Analysis tools Library DNA purification DNA barcoding

21 Normal Illumina workflow

22 Video on Sample preparation

23 Simplified protocol

24 Nextera XT sample prep video Manual:

25 Nextera XT tutorial Nextera DNA Sample Prep. Kit: ca. 20 min

26 Nextera XT library workflow

27 Adapters added by PCR Index (barcode)

28 Multiplexing DNA samples Multiplexing 18 E. coli 24 S. aureus Illumina MiSeq system Medium throughput system

29 Multiplexing with Nextera XT

30 Library building

31 Library preparation movies (Login might be required)

32 How many bacteria in a library? genomes around 5-6 Mb - E. coli - Klebsiella - Salmonella 24 genomes around 3 Mb - Enterococcus - Staphylococcus - Campylobacter

33 The MiSeq principle

34 To the MiSeq Illumina MiSeq system Medium throughput system

35 NGS output Huge numbers of small fragments ( bp)

36 Data analysis of WGS data Assembled data Raw reads Single end Paired end De novo Assembly Com. Software Web tool

37 Reference vs. de novo assembly Reference assembly Known genome De novo assembly Smaller fragments (Unknown order)

38 Reference vs. de novo assembly

39 Data analysis of WGS data Assembled data Species confirmation MLST Resistance genes Virulence genes Plasmids

40 1G bases NGS Illumina PacBio Assembly pipeline 2-6 Mb Allele 1 Allele 2 Resistance Allele 3 MLST gene Outbreak profile strain SNP* Species based ID typing Allele 4 Allele 5 } ST List of genes (100% or >95%) Theoretical resistance phenotype AAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAA Epidemiological Virulence Resistance AAAATAAAAAAAAAAA genes AATAAATAATAATAAA markers *SNP Single Nucleotide Polymorphism (extreme MLST)

41 Data analysis of WGS data Assembled data

42 Data analysis of WGS data Assembled data Species confirmation KmerFinder SpeciesFinder Description Breaks the genome into small 16-mers (k=16) and scans a DB of complete genomes for best match. Identifies 16S in the genome and compare to a database of 16S sequences. Output A sorted list of the number of best-matching 16-mers in a given complete genome (hits in sequence) The best hit. A TRUE value means perfect hit, a FAIL value means close match.

43 Example: KmerFinder

44 Example - KmerFinder

45 Example Resfinder VTEC O104:H4 outbreak strain

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47 For publication in: Journal of Antimicrobial Chemotherapy Genotyping using whole-genome sequencing is a realistic alternative to surveillance based on phenotypic antimicrobial susceptibility testing Ea Zankari 1,2, Henrik Hasman 1, Rolf Sommer Kaas 1,2, Anne Mette Seyfarth 1, Yvonne Agersø 1, Ole Lund 2, Mette Voldby Larsen 2, Frank M. Aarestrup 1,# 200 isolates reduced to 197 (Salmonella, E. coli, E. faecium, E. faecalis) 3,051 individual susceptibility tests

48 Table 2. Overview of resistance genes detected in the isolates by ResFinder with an ID 98.0% Resistance gene No. of isolates (%)* S. Typhimurium (n = 49) E. coli (n = 48) E. faecalis (n = 50) E. faecium (n = 50) Aminoglycoside str 0 (0) 0 (0) 5 (10.0) 1 (2.0) ant(6)-ia 0 (0) 0 (0) 18 (36.0) 18 (36.0) ant(6')-ii 0 (0) 0 (0) 0 (0) 49 (98.0) aph(3')-ia 2 (4.1) 2 (4.2) 0 (0) 0 (0) aph(3')-ic 2 (4.1) 0 (0) 0 (0) 0 (0) aph(3')-iii 0 (0) 0 (0) 17 (34.0) 10 (20.0) aac(6')-aph(2'') 0 (0) 0 (0) 10 (20.0) 0 (0) stra/strb 19 (38.8) 10 (20.8) 0 (0) 0 (0) aada1 5 (10.2) 19 (39.6) 0 (0) 0 (0) aada2 2 (4.1) 4 (8.3) 0 (0) 0 (0) Gene blatem-1 blactx-m-14 blacarb-2 Salmonella 21 (42.9%) 0 (0%) 1 (2.1%) E. Coli 12 (25.0%) 1 (2.1%) 2 (4.1%) aada4 0 (0) 1 (2.1) 0 (0) 0 (0) aada13 1 (2.0) 0 (0) 0 (0) 0 (0) Beta-lactam pbp5 0 (0) 0 (0) 0 (0) 49 (98.0) blatem-1 21 (42.9) 12 (25.0) 0 (0) 0 (0) blatem (0) 1 (2.1) 0 (0) 0 (0) blactx-m-14 0 (0) 1 (2.1) 0 (0) 0 (0) blacarb-2 2 (4.1) 0 (0) 0 (0) 0 (0) MLS erm(b) 0 (0) 1 (2.1) 25 (50.0) 15 (30.0) Isa(A) 0 (0) 0 (0) 50 (100.0) 0 (0) Inu(B) 0 (0) 0 (0) 11 (22.0) 15 (30.0) msr(c) 0 (0) 0 (0) 0 (0) 44 (88.0) mph(a) 0 (0) 1 (2.1) 0 (0) 0 (0) Phenicol cata1 0 (0) 2 (4.2) 0 (0) 0 (0) flor 2 (4.1) 0 (0) 0 (0) 0 (0) cmla1 0 (0) 3 (6.3) 0 (0) 0 (0) cat(pc194) 0 (0) 0 (0) 0 (0) 1 (2.0) Sulphonamide sul1 9 (18.4) 8 (16.7) 0 (0) 0 (0) sul2 20 (40.8) 7 (14.6) 0 (0) 0 (0) sul3 0 (0) 3 (6.3) 0 (0) 0 (0) Tetracycline tet(a) 1 (2.0) 11 (22.9) 0 (0) 0 (0) tet(b) 19 (38.8) 4 (8.3) 0 (0) 0 (0) tet(g) 2 (4.1) 0 (0) 0 (0) 0 (0) tet(m) 0 (0) 0 (0) 34 (68.0) 27 (54.0) tet(l) 0 (0) 0 (0) 24 (48.0) 5 (10.0) tet(s) 0 (0) 0 (0) 0 (0) 1 (2.0) tet(o) 0 (0) 0 (0) 0 (0) 1 (2.0) Trimethoprim dfra1 1 (2.0) 9 (18.8) 0 (0) 0 (0) dfra12 0 (0) 2 (4.2) 0 (0) 0 (0) dfra14 1 (2.0) 0 (0) 0 (0) 0 (0) dfra21 0 (0) 1 (2.1) 0 (0) 0 (0) dfrd 0 (0) 0 (0) 0 (0) 1 (2.0) dfrg 0 (0) 0 (0) 17 (34.0) 0 (0) Glycopeptide Van-A 0 (0) 0 (0) 0 (0) 1 (2.0) MLS, Macrolide-Lincosamide-StreptograminB, *, Per cent resistance genes was determined by dividing the number of isolates harbouring the gene by the total number of isolates (per species).

49 Phenotypic Resistant Susceptible Predicted resistant Predicted susceptible % concordance retest Phenotypic Resistant Susceptible Resistant Susceptible % concordance Spectinomycin in E. coli

50 Example: VirulenceFinder

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52 Plasmid markers

53 Assembled genome/contigs Gram negative plasmids 100% 454 single end reads 454 paired end reads Illumina Gram 98% positive single end plasmids reads Illumina 95% paired end reads Ion Torrent 90% SOLiD single end reads 85% SOLiD paired end reads SOLiD mate pair reads incf plasmid

54 PlasmidFinder

55 Workflow with WGS at the clinical laboratory 4-6 hours Modified from Didelot et al., 2012.

56 E. coli in Urine samples A: ATCC 8739 reference SNP tree ST597 ST597 ST409 ST409 ST227 ST227 _d = Direct sequencing on urine _i = sequencing of isolate from urine

57 Strain KmerFinder SpeciesFinder ResFinder VirulenceFinder PlasmidFinder C751 24_ E64 Skejby2