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Sample to Insight
High resolution outbreak tracing and resistance detection using
whole genome sequencing in the case of a...
Sample to Insight
Legal disclaimer
QIAGEN products shown here are intended for molecular biology
applications. These produ...
Sample to Insight
Outline
Introduction to Mycobacterium tuberculosis1
A case study2
Outbreak tracing3
Detecting antimicrob...
Sample to Insight
Mycobacterium tuberculosis – Infection
Tuberculosis (TB) is one of the deadliest diseases worldwide
• On...
Sample to Insight
Mycobacterium tuberculosis – Genetics
M. tuberculosis strains are highly clonal
• They have a nucleotide...
Sample to Insight
Combatting the TB epidemic
6
Classic methods
New methods:
focus on whole
genome
• Contact investigation
...
Sample to Insight
Combatting the TB epidemic
Challenges
in whole
genome
sequencing
Transmission
at maximum
resolution
Link...
Sample to Insight
Microbial Genomics Pro Suite
Features overview
Microbiome profiling
• 16S and whole metagenome
• Taxonom...
Sample to Insight
Outline
Introduction to Mycobacterium tuberculosis1
A case study2
Outbreak tracing3
Detecting antimicrob...
Sample to Insight
Case study
• Fiebig and co-workers, 2017: Surveillance and outbreak report
• Describes the investigation...
Sample to Insight
Case study
Five MDR-TB VNTR A
IV, V – Diagnosed
in June 2013
I, II, III – Diagnosed
between 2010–12
Born...
Sample to Insight
Case study
Patient ID Sex Age group
(years)
Country of
residence
Country of
birth
Month and year
of curr...
Sample to Insight
Outline
Introduction to Mycobacterium tuberculosis1
A case study2
Outbreak tracing3
Detecting antimicrob...
Sample to Insight
Outbreak tracing
Whole genome sequencing for transmission analysis
WGS for transmission analysis
• Bacte...
Sample to Insight
Outbreak tracing
Automated workflows for outbreak tracing
• Preconfigured workflow “map to
specified ref...
Sample to Insight
Case study results
Outbreak tracing
• Analysis is based on 673 variant positions
High resolution outbrea...
Sample to Insight
Case study results
398
9
4
1
46
34
28
22
6
12
20112012
2011
2014
2010
2004
2011
2013
2014
2011
2013
2011...
Sample to Insight
Outline
Introduction to Mycobacterium tuberculosis1
A case study2
Outbreak tracing3
Detecting antimicrob...
Sample to Insight
Detecting antimicrobial resistance
Mutation detection
• SNVs and InDels
• Local realignment
◦ Reduced FP...
Sample to Insight
Results
Antimicrobial resistance
• Detected resistance causing variants
• Total 123 variants
• 75 from o...
Sample to Insight
Evaluating unknown mutations
Link variants to 3D protein structure (novel variants not found in literatu...
Sample to Insight
Variants in the context of drug-target 3D structure
Link variants to 3D protein structure
DNA gyrase
• T...
Sample to Insight
Variants in the context of drug-target 3D structure
Link variants to 3D protein structure
RNA polymerase...
Sample to Insight
Variants in the context of drug-target 3D structure
More examples
RpoB KatG RpsL PncA
GyrA PncA ThyA Rpo...
Sample to Insight
Summary
• Outbreak tracing
◦ High resolution outbreak analysis
◦ Preconfigured workflows for data QC, ma...
Sample to Insight
Questions?
Contact QIAGEN technical service
Call: 1-800-426-8157 for US
Call: +49 2103-29-12400 EU
Email...
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High Resolution Outbreak Tracing and Resistance Detection using Whole Genome Sequencing in the case of a Mycobacterium Tuberculosis Outbreak

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In March 2014, a molecular cluster of five multidrug-resistant Mycobacterium tuberculosis was detected by the Austrian National Reference Laboratory. An investigation was initiated to determine if transmission had occurred within Austria. Epidemiological links to Germany and Romania prompted a multi-national joint investigation, tracing the outbreak. The results were published by Fiebig and coworkers in 2017.
Whole genome SNP analysis allowed for high resolution clustering of isolates. Whole genome sequencing further permitted simultaneous detection of resistance-causing variants. Using an improved variant detection pipeline, we identified novel variants undetected in the original study. We used functional analysis to explore if novel variants could be associated to antimicrobial resistance.
Using the data published by Fiebig at al. in 2017, we demonstrate the use of CLC Microbial Genomics Module for tracing pathogen transmission during outbreaks and for the detection and functional analysis of resistance-causing variants. The applied user-friendly tools and preconfigured workflows ensure ease of use and reproducibility.

Published in: Health & Medicine
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High Resolution Outbreak Tracing and Resistance Detection using Whole Genome Sequencing in the case of a Mycobacterium Tuberculosis Outbreak

  1. 1. Sample to Insight High resolution outbreak tracing and resistance detection using whole genome sequencing in the case of a Mycobacterium tuberculosis outbreak Winnie Ridderberg, Ph.D. Senior Scientist, Microbial Genomics High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 1
  2. 2. Sample to Insight Legal disclaimer QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of a disease. For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor. High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 2
  3. 3. Sample to Insight Outline Introduction to Mycobacterium tuberculosis1 A case study2 Outbreak tracing3 Detecting antimicrobial resistance4 High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 3
  4. 4. Sample to Insight Mycobacterium tuberculosis – Infection Tuberculosis (TB) is one of the deadliest diseases worldwide • One third of the world’s population has been infected with Mycobacterium tuberculosis • In 2015, 10.4 million became sick with TB and 1.8 million died from TB-related causes worldwide Clinical manifestations of tuberculosis • Pulmonary TB • Extrapulmonary TB affecting organs other than the lungs ◦ Pleura, lymph nodes, abdomen, skin, bones or meninges • Latent TB infection or TB disease Antimicrobial resistance • Multi-drug resistance (MDR) ◦ Resistance to at least isoniazid and rifampicin • Extensive drug resistance (XDR) ◦ MDR plus resistance to a fluoroquinolone and an injectable antibiotic (amikacin, kanamycin, or capreomycin) High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 4
  5. 5. Sample to Insight Mycobacterium tuberculosis – Genetics M. tuberculosis strains are highly clonal • They have a nucleotide identity of 99.9% • They display very little recombination • The main mechanism of antimicrobial resistance is acquiring mutations affecting binding pocket and drug-target interactions High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 5
  6. 6. Sample to Insight Combatting the TB epidemic 6 Classic methods New methods: focus on whole genome • Contact investigation • Phenotypic methods • Molecular methods ◦ Spoligo-typing ◦ 24-MIRU-VNTR • Antimicrobial susceptibility testing • Core-genome MLST www.cgmlst.org • NGS–based whole genome analysis • To control the spread of TB, particularly MDR/XDR-TB, surveillance is key • For correct and effective treatment, understanding the resistance mechanisms is vital High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak
  7. 7. Sample to Insight Combatting the TB epidemic Challenges in whole genome sequencing Transmission at maximum resolution Linking mutations to resistance Accurate detection of resistance mutations Evaluating unknown mutations High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 7
  8. 8. Sample to Insight Microbial Genomics Pro Suite Features overview Microbiome profiling • 16S and whole metagenome • Taxonomic profiling • Functional profiling • Comparative analytics NGS-based isolate typing • Taxonomy • AM resistance • Epidemiology • Molecular phylogeny Bx De Novo Assembly • Genome and metagenome • PacBio • Gene finding • Annotation Pathogen typing AMR typing Epidemiolgy Food safety Microbiome analysis High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 8
  9. 9. Sample to Insight Outline Introduction to Mycobacterium tuberculosis1 A case study2 Outbreak tracing3 Detecting antimicrobial resistance4 High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 9
  10. 10. Sample to Insight Case study • Fiebig and co-workers, 2017: Surveillance and outbreak report • Describes the investigation of a molecular cluster of multi-drug-resistant TB across Austria, Germany and Romania Fiebig, L., Kohl, T.A., Popovici, O., Mühlenfeld, M., Indra, A., Homorodean, D.,Chiotan, D., Richter, E., Rüsch-Gerdes, S., Schmidgruber, B., Beckert, P., Hauer, B.,Niemann, S., Allerberger, F. and Haas, W (2017) A joint cross-border investigation of a cluster of multidrug-resistant tuberculosis in Austria, Romania and Germany in 2014 using classic, genotyping and whole genome sequencing methods: lessons learnt. Euro Surveill. 22,1–11 High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 10
  11. 11. Sample to Insight Case study Five MDR-TB VNTR A IV, V – Diagnosed in June 2013 I, II, III – Diagnosed between 2010–12 Born in Austria Originate from the same city in Romania Live in city 1 I, II – Moved to city 1 III – Moved to city 2 III had a sister with MDR-TB in Germany Austria Three MDR-TB VNTR A VI, VII, VIII – Diagnosed in 2011 VI – Sister of III VII Originate from Romania Born in Africa Germany VIII Five MDR-TB analyzed IX, X, XI, XII, XIII – Diagnosed between 2004–14 X, XI – VNTR A IX – VNTR B Born and live in Romania Romania XII, XIII – VNTR C Five patients originate from the same city in Romania I, II, III, VI, VII High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 11
  12. 12. Sample to Insight Case study Patient ID Sex Age group (years) Country of residence Country of birth Month and year of current TB diagnosis Year of previous TB diagnosis Site of disease I Female 30–39 Austria Romania March 2010 2001 Pulmonary II Male 50–59 Austria Romania January 2011 Pulmonary III Female 30–39 Austria Romania March 2012 1998 and 2003 Pulmonary IV Male 40–49 Austria Austria June 2013 Pulmonary V Male 50–59 Austria Austria June 2013 Pulmonary VI Female 30–39 Germany Romania December 2011 Pulmonary VII Female 30–39 Germany Romania May 2011 Pulmonary VIII Male 30–39 Germany Nigeria July 2011 Extrapulmonary IX Male 40–49 Romania Romania January 2004 Pulmonary X Male 50–59 Romania Romania December 2011 2011 Pulmonary XI Male 30–39 Romania Romania January 2014 Pulmonary XII Male 20–29 Romania Romania December 2013 Pulmonary XIII Female 60–69 Romania Romania January 2014 2004 Pulmonary High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 12
  13. 13. Sample to Insight Outline Introduction to Mycobacterium tuberculosis1 A case study2 Outbreak tracing3 Detecting antimicrobial resistance4 High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 13
  14. 14. Sample to Insight Outbreak tracing Whole genome sequencing for transmission analysis WGS for transmission analysis • Bacteria evolve throughout the progression of an outbreak ◦ Genome-wide comparison of mutations offers highest resolution for clustering and source tracking • Methods ◦ Genome-wide variant (SNP, SNVs) detection ◦ Phylogenetic analysis via SNP trees Whole genome sequencing Variant detection Phylogenetic analysis High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 14
  15. 15. Sample to Insight Outbreak tracing Automated workflows for outbreak tracing • Preconfigured workflow “map to specified reference” • Output from the variant detection tool was used for SNP-tree calculation High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 15
  16. 16. Sample to Insight Case study results Outbreak tracing • Analysis is based on 673 variant positions High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 16
  17. 17. Sample to Insight Case study results 398 9 4 1 46 34 28 22 6 12 20112012 2011 2014 2010 2004 2011 2013 2014 2011 2013 2011 2013 VNTR “C” VNTR “B” VNTR “A” Both born and living in Romania All originate from the same Romanian city, but live in three countries Sisters Three patients from the same Austrian city Whole genome sequencing and SNP analysis provide higher resolution outbreak investigation compared to genotyping methods. High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 17
  18. 18. Sample to Insight Outline Introduction to Mycobacterium tuberculosis1 A case study2 Outbreak tracing3 Detecting antimicrobial resistance4 High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 18
  19. 19. Sample to Insight Detecting antimicrobial resistance Mutation detection • SNVs and InDels • Local realignment ◦ Reduced FP InDel calling • Low Frequency Variant Detection tool • Optional: further variant filtering after variant detection ◦ Reduced FN rate Reference database • 1459 variants (SNPs and InDels) • 31 loci • 15 drugs Sources for variant database • Coll et al. (2015) ◦ TBDreaMDB ◦ MUBII–TB–DB ◦ Recent literature to include additional variants and loci • Additional variants described by Miotto et al. (2014) and Allana et al. (2017) High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 19
  20. 20. Sample to Insight Results Antimicrobial resistance • Detected resistance causing variants • Total 123 variants • 75 from original study • 48 new How do you evaluate novel variant calls (missing in the database)? High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 20 Original study New analysis No. of variants % No. of variants % In congruence with phenotype 61 81 30 63 Antimicrobials not tested 8 11 14 29 In contrast with phenotype 6 8 4 8 Total 75 48 Drug Locus No. of variants detected in paper No. of additional detected variants Capreomycin tlyA 0 2 Capreomycin rrs 2 4 Clofazimide RV0678 0 1 Ethambutol embB 15 1 Ethambutol embC 0 1 Ethambutol embR 0 2 Ethionamide ethA 5 0 Ethionamide fabG1 promoter 0 10 Fluoroquinolones gyrA 1 1 Fluoroquinolones gyrB 0 1 Isoniazid fabG1 promoter 0 10 Isoniazid katG 13 5 Kanamycin eis promoter 0 6 Kanamycin rrs 2 4 Para-aminosalisylic acid thyA 1 0 Pyrazinamide pncA 11 8 Rifampicin rpoB 14 2 Rifampicin rpoC 0 4 Streptomycin gidB 11 0 Streptomycin rpsL 2 0 Streptomycin rrs 2 4
  21. 21. Sample to Insight Evaluating unknown mutations Link variants to 3D protein structure (novel variants not found in literature) • Visualization of amino acid alterations on 3D models • Evaluate the effect of variants on the drug-target interaction or protein function High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 21
  22. 22. Sample to Insight Variants in the context of drug-target 3D structure Link variants to 3D protein structure DNA gyrase • Target of fluoroquinolones • Involved in supercoiling of DNA, binding DNA and introducing transient double- stranded breaks • Fluoroquinolones bind to the enzyme-DNA complex • Mutation of the target region alter target structure and the binding affinity for the drug DNA Gyrase Subunit A Moxifloxacin Reference model Variant model - Ala90Val GyrA High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 22
  23. 23. Sample to Insight Variants in the context of drug-target 3D structure Link variants to 3D protein structure RNA polymerase • Target of rifamycin • Rifamycin binds within the DNA/RNA channel physically blocking elongation • Resistance arises from amino acid alterations in the binding site, decreasing the affinity for the drug • Sensitive variant detection reveals more mutations with likely causal effects. • 3D structure to assess impact of mutations on drug-target interaction. Reference model Variant model – Ser431Asn RNA Polymerase Subunit B Rifampicin RpoB High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 23
  24. 24. Sample to Insight Variants in the context of drug-target 3D structure More examples RpoB KatG RpsL PncA GyrA PncA ThyA RpoC KatG TlyA EmbR Rv0678 High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 24
  25. 25. Sample to Insight Summary • Outbreak tracing ◦ High resolution outbreak analysis ◦ Preconfigured workflows for data QC, mapping and variant detection ◦ SNP tree for visualization of outbreak isolates and associated metadata • Resistance detection ◦ Optimized variant detection ◦ Filtering against known resistance causing variants ◦ 3D visualization for qualification of undescribed variants High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 25
  26. 26. Sample to Insight Questions? Contact QIAGEN technical service Call: 1-800-426-8157 for US Call: +49 2103-29-12400 EU Email: techservice-na@QIAGEN.com techservice-eu@QIAGEN.com QIAwebinars@QIAGEN.com Thank you for attending! High resolution outbreak tracing and resistance detection using WGS in the case of a M. tuberculosis outbreak 26

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