This document summarizes bioinformatics tools that can be used for analysis of high-throughput sequencing data for molecular diagnostics. It discusses databases for virulence factors and antimicrobial resistance as well as tools for assembly, annotation, pan-genome analysis, visualization, and commercial solutions. The presentation emphasizes that there is no single best tool and different approaches are needed for different questions. Collaboration with other researchers is recommended.

1. João André Carriço,
Microbiology Institute and Instituto de Medicina Molecular,
Faculty of Medicine, University of Lisbon
jcarrico@fm.ul.pt twitter: @jacarrico
ME081 – Meet-The-Expert Session
26th ECCMID, Amsterdam, Netherlands
7-12 April 2016

2.  This presentation is not intended to cover all available
software or databases (we would need several weeks or
months to do that)
 I’ll present what I use or intend to use in a near future
 I gladly accept any suggestions to included on similar
presentations in the future.
 It is supposed to be interactive so ask away during the
presentation.

3.  Available Databases
 Virulence Factors and AMR DBs
 Sequence-based typing databases: Pubmlst.org / Enterobase
 HighThroughput Sequencing data analysis (freeware)
 Prokka
 Roary
 Nullabor
 Microreact.org
 PHYLOViZ
 Commercial Solutions
 Bionumerics 7.5
 CLC GenomicsWorkbench (CLC Bio)
 Ridom Seqsphere+

4. Virulence Factor Databases
 VFDB (http://www.mgc.ac.cn/VFs/main.htm)
 Pathosystems Resource Integration Center (PATRIC)
VF (https)://www.patricbrc.org/)
 Victors (http://www.phidias.us/victors/)
 PHI-Base (http://www.phi-base.org/)
 MvirDB (http://mvirdb.llnl.gov/ )
To know more:
- Presentation on the Controversies in interpreting whole genome sequence data session :
http://eccmidlive.org/#resources/how-can-we-design-actionable-virulome-databases

5.  Comprehensive Antibiotic Resistance Database
(CARD) (https://card.mcmaster.ca/)
 Repository of Antibiotic resistanceCassetes
(RAC)(http://rac.aihi.mq.edu.au/rac/)
 Integrall :The integron database
(http://integrall.bio.ua.pt/)
(…)

6. To know more :
http://www.slideshare.net/nickloman/eccmid-2015-so-i-have-sequenced-my-genome-what-now
Reads
(fastq files)
contigs
(fasta files)
Annotated contigs
(gbk/gff files)
Roary :PanGenome Analysis
Enterobase
BIGSdb
Nullabor
PHYLOViZ:
Tree + metada
visualization
Microreact.org:
Tree +metadata
+vizualization
Prokka
De novo assembler

7. http://www.pubmlst.org
http://bigsdb.web.pasteur.fr/

8. slide by @happy_khan
Martin Sergeant
Mark Achtman
Nabil-Fareed Alikhan
Zhemin Zhou

9.  Genome annotation made easy byTorsten
Seemann (slides byTorsten)
 Genome annotation: adding biological
information to the sequence, by describing
features
To know more :
http://www.slideshare.net/torstenseemann/prokka-rapid-bacterial-genome-annotation-abphm-2013
Available at: https://github.com/tseemann/prokka

10.  Pan genome analysis by Andrew Page
 Available at: https://sangerpathogens.github.io/Roary/
Core
genome
Accessory
genome
Pan-genome

11.  Inputs:Annotated de novo assemblies (GFF files)
• Typically from the annotation pipeline
 Outputs:
• Spreadsheet with presence and absence of genes
• Multi-FASTA alignment of core genes so you can build a tree
without a reference
• Multi-FASTA alignments for each gene
• Plots for the open/closed genome, unique genes
• Integrates with iCANDY so you can visualise all structural variation
• QC report from Kraken to help identify suspect samples
(Slide by Andrew Page)

12. Core (n or n-1 strains)
Soft-Core
(n-2 or n-3 strains)
Shell
( 8(?) to n-3 strains)
Cloud
( <8 (?) strains)
Core genome:
Core + Soft-Core
Accessory genome:
Shell + Cloud

13. iCANDY output of presence and
absence of genes in accessory
genome.
S. Weltevreden & public S. enterica
genomes
(Slide by Andrew Page)

14.  Complete pipeline from reads to reports byTorsten
Seemann
 Objective is automate analysis for everyday use on
public health labs /research settings
 Uses and distills outputs by a lot of software
 Avaliable at: https://github.com/tseemann/nullarbor

15. Slide byTorsten Seeman

16. From: https://github.com/tseemann/nullarbor

17. Slides byTorsten Seeman

18. www.phyloviz.net

19. Inputs:
- Tab separated txt (profiles)
- Fasta files
- Automatic database retrieval
(MLST)
Outputs:
• goeBURST and goeBURST
MST
• Link quality assessment
• High quality images
Can be easily applied to:
- MLST/ cgMLST/wgMLST
- MLVA
- SNP data*
- Gene Presence/absence

20. New features:
• Hierarchical clustering
• Neighbor-Joining
• Project Saving

21.  Available at http://online.phyloviz.net
 Web based version of PHYLOViZ
 Allows users to create their own datasets, save them and share their data
(privately or publicly)
 REST API available
 Scalable to thousands of nodes
 Tree Analysis tools:
 Interactive distance matrix
 NLV graph

22. Slide by @happy_khan

24. NLV Graph
Tree cut-off
Full MST

27. Create Selections
Change tree options

28.  Available at http://microreact.org/
 Presentation on session Harnessing whole genome sequence data
for public health applications : Novel open access tools forWGS-
based pathogen surveillance and the identification of high-risk
clones
 http://eccmidlive.org/#resources/novel-open-access-tools-for-
wgs-based-pathogen-surveillance-and-the-identification-of-high-
risk-clones

30. • Ridom Seqsphere+ : http://www.ridom.de/seqsphere/
• Applied Maths Bionumerics 7.6: http://www.applied-maths.com/bionumerics
• CLCBioGenomicWorkbench : http://www.clcbio.com/blog/clc-genomics-workbench-7-5/

31. • Huge variety of software and database solutions
• There is no single One-Size-Fits-All solution (job
security for bioinformaticians)
• Different questions require different approaches
• Always questions the results and data provenance

32.  ECCMID2015 Meet-the-expert session on “What bioinformatic tools
should I use for analysis of HighThroughput Sequencing data for
molecular diagnostics? ”
 Nick Loman: http://www.slideshare.net/nickloman/eccmid-2015-
meettheexpert-bioinformatics-tools
 João André Carriço:
http://www.slideshare.net/joaoandrecarrico/eccmid-meet-
theexpert2015

33.  UMMI Members
 Bruno Gonçalves
 Mário Ramirez
 José Melo-Cristino
 INESC-ID
 Alexandre Francisco
 Cátia Vaz
 Marta Nascimento
 EFSA INNUENDO Project (https://sites.google.com/site/innuendocon/)
 Mirko Rossi
 FP7 PathoNGenTrace (http://www.patho-ngen-trace.eu/):
 Dag Harmsen (Univ. Muenster)
 Stefan Niemann (Research Center Borstel)
 Keith Jolley, James Bray and Martin Maiden (Univ. Oxford)
 Joerg Rothganger (RIDOM)
 Hannes Pouseele (Applied Maths)
 Genome Canada IRIDA project (www.irida.ca)
 Franklin Bristow, Thomas Matthews, Aaron Petkau, Morag Graham and Gary Van Domselaar(NLM , PHAC)
 Ed Taboada and Peter Kruczkiewicz (LabFoodborne Zoonoses, PHAC)
 Fiona Brinkman (SFU)
 William Hsiao (BCCDC)
INTEGRATED RAPID INFECTIOUS DISEASE ANALYSIS