MIRU-VNTRplus: a web tool for polyphasic
genotyping of Mycobacterium tuberculosis
complex bacteria
Thomas Weniger1,*, Justina Krawczyk2, Philip Supply3, Stefan Niemann2 and
Dag Harmsen1
1Department of Periodontology, University Hospital Mu¨nster, Mu¨nster, 2Molecular Mycobacteriology,
Forschungszentrum Borstel, Borstel, Germany and 3CIIL – Center for Infection and Immunity of Lille
INSERM U 1019, CNRS UMR 8204 Univ Lille Nord de France Institut Pasteur de Lille, France
Received January 28, 2010; Revised April 15, 2010; Accepted April 22, 2010
ABSTRACT
Harmonized typing of bacteria and easy identifica-
tion of locally or internationally circulating clones
are essential for epidemiological surveillance and
disease control. For Mycobacterium tuberculosis
complex (MTBC) species, multi-locus variable
number tandem repeat analysis (MLVA) targeting
mycobacterial interspersed repetitive units (MIRU)
has been internationally adopted as the new
standard, portable, reproducible and discriminatory
typing method. However, no specialized bioinfor-
matics web tools are available for analysing MLVA
data in combination with other, complementary
typing data. Therefore, we have developed the web
application MIRU-VNTRplus (http://www.miru-
vntrplus.org). This freely accessible service allows
users to analyse genotyping data of their strains
alone or in comparison with a reference database
of strains representing the major MTBC lineages.
Analysis and comparisons of genotypes can be
based on MLVA-, spoligotype-, large sequence poly-
morphism and single nucleotide polymorphism
data, or on a weighted combination of these
markers. Tools for data exploration include search
for similar strains, creation of phylogenetic and
minimum spanning trees and mapping of geograph-
ic information. To facilitate scientific communica-
tion, an expanding genotype nomenclature (MLVA
MtbC15-9 type) that can be queried via a web- or a
SOAP-interface has been implemented. An exten-
sive documentation guides users through all appli-
cation functions.
INTRODUCTION
The bacteria of the Mycobacterium tuberculosis complex
(MTBC) are the causative agents of tuberculosis (TB).
This infectious disease is responsible for approximately 2
million deaths annually and foci of multi- and extensive
drug resistance are emerging worldwide (1). Genotyping
of MTBC strains empowers epidemiological surveillance
and control, e.g. by permitting detection of clonal spread
of multi-drug resistant strains and unsuspected outbreaks.
At the clinical level, molecular typing enables iden-
tification of false positive cases due to laboratory cross-
contamination and distinction between exogenous reinfec-
tion and relapse from initial infection. From a research
perspective, molecular strain typing is valuable for the de-
ciphering of the MTBC population structure and thus
enabling the development of new diagnostics, vaccines
or treatments (2).
The previous gold standard for MTBC genotyping—
IS6110 restriction fragment length polymorphism
(RFLP) typing—is laborious and time consuming and
the resulting complex banding patterns make inter-
laboratory comparisons difficult (3). As the MTBC gene
sequence variation is low classical sequence-based typing
methods such as multi-locus sequence typing, even
extended to tens of genes, are only informative for the
identification at genetic (sub-)lineage but not at strain
level (4,5). Likewise, large sequence polymorphisms
(LSP) or single nucleotide polymorphisms (SNP)
elucidated MTBC genetic lineages differing in their geo-
graphical distribution immunogenicity, virulence and as-
sociation with multidrug resistant TB (6–8).
However, the use of other, more variable markers is
needed for a desirable finer phylogenetic classification
and detection down to strain level to the advantage of
public health and clinical objectives. As a portable,
*To whom correspondence should be addressed. Tel: +49 251 83 49882; Fax: +49 251 83 47134; Email: tweniger@uni-muenster.de
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
W326–W331 Nucleic Acids Research, 2010, Vol. 38, Web Server issue Published online 10 May 2010
doi:10.1093/nar/gkq351
� The Author(s) 2010. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
reproducible and discriminatory typing tool, the
multi-locus variable number tandem repeat (VNTR)
analysis (MLVA) targeting a number of tandem repeat
loci including the mycobacterial interspersed repetitive
units (MIRU) has, therefore, been internationally
adopted as the new standard (9,10). Spoligotyping is
often used as a rapid additional typing method to
increase the discriminatory power of MIRU-VNTR
typing. This method addresses the presence or absence
of 43 spacers at the so-called direct repeat locus, a para-
digmatic member of the family of clustered regularly
interspaced short palindromic repeats (CRISPR) (11,12).
In addition to their combined discriminatory power,
24-locus-based MIRU-VNTR typing and spoligotyping
data are predictive of the main MTBC phylogenetic
lineages, although they are somewhat less deterministic
for such purposes than are LSP or sequence-based
data (13,14).
Several web services containing strain collections
of MLVA or spoligotyping data are available, e.g.
MLVAbank or spolTools and SITVIT/SpolDB4 (15–18).
Each of these web applications offers functions for simple
data comparison and basic clustering for only one
genotyping method. However, there was no online tool
available that provided a comprehensive analysis of
MTBC strains based on multiple genotyping methods
and tools for directly naming new genotypes. We
described recently such a system, i.e. MIRU-VNTRplus,
with a focus on evaluation of a reference database that
allows a robust-lineage identification based on the com-
bination of different genotyping data (19). Here, we
describe for the first time the complete functionality of
MIRU-VNTRplus, including the new features minimum
spanning tree (MST), geographic mapping and the no-
menclature service.
IMPLEMENTATION
The MIRU-VNTRplus server was implemented in Java
using the JavaServer Faces technology together with the
RichFaces AJAX extension for creating the web pages.
Java Servlets were used for image rendering. In order to
store the data of the provided nomenclature service per-
sistently, a MySQL database was used. Database access
was implemented by means of the object-relational
mapping framework Hibernate, while the SOAP interface
for accessing the nomenclature service was realized by
means of the Apache Axis2 framework. The mapping
function uses Google Maps for geocoding and display of
an interactive map. MIRU-VNTRplus is compatible with
the most common web browsers, i.e. Mozilla Firefox 2
and 3; Microsoft Internet Explorer 6, 7 and 8; Opera 9
and 10; Apple Safari 4; and Google Chrome 3. To enable
the application’s full functional capability JavaScript has
to be activated.
The application allows the input of copy numbers for 24
MIRU-VNTR loci and 15 or 12 loci subsets (10).
Furthermore, the following typing data are supported:
presence or absence of 43 spoligotyping spacers,
presence or absence of 15 standard LSPs (6,7), values of
six SNPs (20) and susceptibility data for 16 drugs. Finally,
in addition to general descriptive information (e.g. strain
ID and country of isolation) input for three user-specific
data fields can be supplied. Furthermore, a reference
database containing gentoyping data of 186 strains repre-
senting the major MTBC lineages is available (19). IS6110
RFLP fingerprinting images are also shown for visual
comparison of these strains.
Distance measures that can be used for strain compari-
son include categorical distance, DC, (dm)2 and DSW
(21–23). Double and variant alleles are regarded as own
categories for categorical distance calculation and are
treated as missing data for all other distance measures.
When used for pairwise strain comparisons, the categor-
ical distance is identical to the distance measure DA that is
well suited for phylogenetic analysis of VNTR markers
(24). Polyphasic typing is achieved by calculating
combined pairwise distances for all chosen typing
methods. A combined distance is calculated by multiply-
ing the distance of each method by a weighting factor,
summing up the distances for all methods, and dividing
this value by the sum of the weighting factors. Missing
data is ignored for the pairwise distance calculation,
thus allowing users to work with a self-defined subset of
loci.
Phylogenetic trees are inferred using the un-weighted
pair group method with arithmetic means (UPGMA) or
neighbour-joining (NJ) algorithms (25,26). Generated
trees can be re-rooted using a manually selected
outgroup. The resulting trees can be exported to Newick
and NEXUS format and the underlying distance matrix
can be downloaded as a MEGA file. A minimum spanning
tree is created using Kruskal’s algorithm and a
force-directed graph layout for visualization (27). All
trees can be downloaded as raster image (PNG), vector
image (SVG, EMF) or PDF document.
RESULTS
The freely accessible MIRU-VNTRplus web application
(http://www.miru-vntrplus.org) offers three main func-
tions: (i) phylogenetic lineage identification by using a
reference database; (ii) analysis and visualization
of genotyping data; and (iii) access to the MLVA
MtbC15-9 nomenclature service (Figure 1). An extensive
documentation including a manual, multi-media tutorials
and protocols for the genotyping methods complements
the web server.
Data input
Genotyping data can be entered for a single strain via a
web form, copied from a spreadsheet application via clip-
board or uploaded by using Comma Separated Values
(CSV) or Microsoft (MS) Excel files. Template CSV and
MS Excel files that simplify the upload process are avail-
able. MIRU-VNTRplus allows users to upload data for
up to 500 strains. Various formats for genotyping data are
accepted, e.g. MIRU-VNTR alleles with incomplete
tandem repeat units (variant alleles) and two alleles sim-
ultaneously detected for a given locus (double alleles),
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CDC notation for MIRU-VNTR data (by using ‘A’ for 10
repeats, ‘B’ for 11 repeats, etc. double digits are avoided)
or binary and octal numbers for spoligotyping patterns.
After exclusion of possible PCR artefacts (i.e. classical
stutter peaks) concordantly observed double alleles in
several independent VNTR loci for a given sample
indicate the presence of a mixed DNA population. This
mixed population can result from a true mixed infection,
or from culture or DNA contamination. In contrast, the
occurrence of double alleles in a single locus rather
suggests the presence of a given allelic variant within a
clonal isolate. In the following, the entered or uploaded
strain data is referred to as ‘user strains’.
Analysis and visualization of genotyping data
MIRU-VNTRplus supports polyphasic typing by
calculating a combined distance for different types of
genotyping data. An input form allows the selection of
the used genotyping methods, distance measures and
weightings (Figure 2A). This form can be accessed on all
pages that use calculated distances. A data table that can
be sorted by any column displays the strain data. Using
the UPGMA or NJ algorithm a dendrogram can be
directly included in this table, thus ordering the strains
by their position in the tree (Figure 2B). A filter
function allows one to exclude strains from analysis that
do not match certain criteria. Complex filtering criteria
can be created by combining comparison operators for
certain data fields with the logical operators AND or
OR. Strains can be marked manually or automatically
with a background colour, e.g. according to the value of
lineage, genetic marker or user data. By selecting a strain
as the comparison strain all genetic marker differences are
highlighted for all other strains. In addition the distances
between the comparison strain and all other strains are
displayed for sorting and filtering.
Phylogenetic trees can be created and modified inter-
actively on an extra page. Depending on the user selection,
trees are displayed as dendrograms or radial graphs.
The label text can be chosen and genotyping data can be
added to the image. It is possible to export the distance
matrix, the tree and the resulting image to various
formats. Clicking a branch or leaf of the tree opens a
pop-up window that shows the genotyping data for all
sub-tree strains and offers to re-root the tree, swap
branches and mark sub-tree strains by colour as inter-
active features. In addition to classical phylogenetic
trees, MIRU-VNTRplus also allows the calculation of
MSTs. However, MSTs can be drawn based on data
from only one typing method at a time. Clonal complexes
(CC) as defined by genotypes sharing a selectable
maximum locus difference are highlighted in the MST
image. The choice of the label text, length of connection
lines and zoom factor modifies the appearance of the
MST. Using the context menu strains can again be
marked by colour.
The strain-mapping feature displays the geographical
distribution of strains on a map. The location information
is retrieved from the data field country of isolation that
might as well include ZIP codes or city information. The
distribution of species, lineages or user data field values
for each location is visualized in a colour-coded pie chart.
Phylogenetic lineage identification
The comparison with the reference database allows the
identification of the phylogenetic lineage of user strains.
Again, the identification can be based on a combined
distance of several genotyping methods. Identification by
similarity search lists reference database strains displaying
an adjustable maximum distance to each user strain. The
default maximum distance has been determined on the
basis of validation tests using an external strain data set
(19). The similarity search results can be used for the auto-
matic assignment of species and lineage information to
user strains. All results are exportable as MS Excel or
CSV file. In most cases, identification by similarity
search will not be sufficient to undoubtedly identify all
user strains. Here, an additional tree-based analysis can
be carried out by calculating a UPGMA or NJ tree that
contains all user and reference database strains.
Investigating strains that are monophyletic (i.e.
ingroups) compared to reference database strains in the
different tree branches allow the user to infer species and
lineage assignment. Such assignments may then be set by
clicking strains or nodes on the tree (Figure 3).
MtbC15-9 nomenclature service
For the exchange of MIRU-VNTR data, the reporting of
the full genotype with copy numbers of each locus in
perfect order is mandatory. To facilitate scientific commu-
nication, MIRU-VNTRplus introduces an expanding no-
menclature that assigns a numerical code to MIRU-
VNTR patterns. The MLVA MtbC15-9 type is a juxta-
position of two subtypes, i.e. the MtbC15 and MtbC9
type. These types are based on a set of the 15 most dis-
criminatory MIRU-VNTR loci and a set of nine ancillary
loci, as inferred by the analysis of single locus variation
frequencies on a large international strain collection (10).
The web application provides forms for converting
Figure 1. The three main functions of MIRU-VNTRplus are (i) phylo-
genetic lineage identification by using a reference database; (ii) analysis
and visualization of genotyping data; and (iii) access to the MLVA
MtbC15-9 nomenclature service.
W328 Nucleic Acids Research, 2010, Vol. 38, Web Server issue
carried out by using the menu command ‘Automatically
Set Best Matching Species and Lineage’ with a distance
threshold of 0.17 (default value). By means of the
tree-based identification, lineages of additional user
strains can be derived. By drawing a NJ tree that is
re-rooted at the branch containing the two M. canetti ref-
erence database strains (outgroup), species and lineage in-
formation can be reliably determined for 17 additional
strains (one strain with lineage West African 1, three
strains with lineage S, one strain with Beijing lineage,
eight strains with lineage LAM and four strains with
lineage EAI). A closer look at the remaining unidentified
strains clearly reveals the presence of the two lineages
(strains with ID 19, 25, 35, 40, 52, 42, 63 and ID 33, 78,
83, 56, 34, 72, 50, 51, 12, 79), which had been described as
‘Sierra Leone-1’ and ‘Sierra Leone-2’ in the original pub-
lication. The creation of a MST using the 24
MIRU-VNTR loci and a maximum locus difference of
four within a CC reproduces the CCs of the publication,
however, with a slightly different visualization and
ordering. Remarkably, MST groups the ‘Sierra Leone-1’
and ‘Sierra Leone-2’ strains into specific CCs. The key
findings of Homolka et al. (28) are thus confirmed by
using MIRU-VNTRplus. The data exploration table
gives an overview of all strains and allows further data
exploration. Possible new MtbC15-9 types can be
submitted by choosing the menu command ‘Assign
MLVA MtbC15-9 Types’.
CONCLUSION
The MIRU-VNTRplus application allows quick identifi-
cation of lineages for MTBC strains and exploration of
data based on a combination of up to four different
genotyping methods. Furthermore, an expanding nomen-
clature service for MLVA MtbC types has been estab-
lished. It is planned to extend the reference database
by adding further quality-controlled data sets. Future
developments will include an open, extendable database
with data sets from other researchers that can be used in
addition to the reference database. Since MLVA, CRISPR
and SNP typing schemes are being published for an
increasing number of especially monomorphic pathogenic
bacterial species, MIRU-VNTRplus may serve as a model
for a powerful, generalized tool to analyse genotypes using
these markers and other categorical data.
FUNDING
German Federal Ministry of Education and Research in
the framework of the Network Zoonoses (grant number
01KI07124 to D.H. for development of the SOAP-
interface); PathoGenomikPlus Network (grant number
0313801J to S.N. for collecting strains and genotyping).
Funding for open access charge: German Federal
Ministry of Education and Research (grant number
01KI07124 to D.H.).
Conflict of interest statement: P. Supply has declared a
potential conflict of interest. P. Supply is a consultant
for Genoscreen, Lille, France. All other authors have
declared that no competing interests exist.
REFERENCES
1. World Health Organization. (2009) Global tuberculosis control.
WHO Report. World Health Organization, Geneva, Switzerland.
2. Gagneux,S. and Small,P.M. (2007) Global phylogeography of
Mycobacterium tuberculosis and implications for tuberculosis
product development. Lancet Infect. Dis., 7, 328–337.
3. van Embden,J.D., Cave,M.D., Crawford,J.T., Dale,J.W.,
Eisenach,K.D., Gicquel,B., Hermans,P., Martin,C., McAdam,R.
and Shinnick,T.M. (1993) Strain identification of Mycobacterium
tuberculosis by DNA fingerprinting: recommendations for a
standardized methodology. J. Clin. Microbiol., 31, 406–409.
4. Sreevatsan,S., Pan,X., Stockbauer,K.E., Connell,N.D.,
Kreiswirth,B.N., Whittam,T.S. and Musser,J.M. (1997) Restricted
structural gene polymorphism in the Mycobacterium tuberculosis
complex indicates evolutionarily recent global dissemination.
Proc. Natl Acad. Sci. USA, 94, 9869–9874.
5. Hershberg,R., Lipatov,M., Small,P.M., Sheffer,H., Niemann,S.,
Homolka,S., Roach,J.C., Kremer,K., Petrov,D.A., Feldman,M.W.
et al. (2008) High functional diversity in Mycobacterium
tuberculosis driven by genetic drift and human demography.
PLoS Biol., 6, e311.
6. Brosch,R., Gordon,S.V., Marmiesse,M., Brodin,P., Buchrieser,C.,
Eiglmeier,K., Garnier,T., Gutierrez,C., Hewinson,G., Kremer,K.
et al. (2002) A new evolutionary scenario for the Mycobacterium
tuberculosis complex. Proc. Natl Acad. Sci. USA, 99, 3684–3689.
7. Gagneux,S., DeRiemer,K., Van,T., Kato-Maeda,M., de Jong,B.C.,
Narayanan,S., Nicol,M., Niemann,S., Kremer,K., Gutierrez,M.C.
et al. (2006) Variable host-pathogen compatibility in
Mycobacterium tuberculosis. Proc. Natl Acad. Sci. USA, 103,
2869–2873.
8. Hirsh,A.E., Tsolaki,A.G., DeRiemer,K., Feldman,M.W. and
Small,P.M. (2004) Stable association between strains of
Mycobacterium tuberculosis and their human host populations.
Proc. Natl Acad. Sci. USA, 101, 4871–4876.
9. Supply,P., Magdalena,J., Himpens,S. and Locht,C. (1997)
Identification of novel intergenic repetitive units in a
mycobacterial two-component system operon. Mol. Microbiol., 26,
991–1003.
10. Supply,P., Allix,C., Lesjean,S., Cardoso-Oelemann,M., Ru¨sch-
Gerdes,S., Willery,E., Savine,E., de Haas,P., van Deutekom,H.,
Roring,S. et al. (2006) Proposal for standardization of optimized
mycobacterial interspersed repetitive unit-variable-number tandem
repeat typing of Mycobacterium tuberculosis. J. Clin. Microbiol.,
44, 4498–4510.
11. Kamerbeek,J., Schouls,L., Kolk,A., van Agterveld,M., van
Soolingen,D., Kuijper,S., Bunschoten,A., Molhuizen,H., Shaw,R.,
Goyal,M. et al. (1997) Simultaneous detection and strain
differentiation of Mycobacterium tuberculosis for diagnosis and
epidemiology. J. Clin. Microbiol., 35, 907–914.
12. Oelemann,M.C., Diel,R., Vatin,V., Haas,W., Ru¨sch-Gerdes,S.,
Locht,C., Niemann,S. and Supply,P. (2007) Assessment of an
optimized mycobacterial interspersed repetitive-
unit-variable-number tandem-repeat typing system combined with
spoligotyping for population-based molecular epidemiology
studies of tuberculosis. J. Clin. Microbiol., 45, 691–697.
13. Wirth,T., Hildebrand,F., Allix-Be´guec,C., Wo¨lbeling,F.,
Kubica,T., Kremer,K., van Soolingen,D., Ru¨sch-Gerdes,S.,
Locht,C., Brisse,S. et al. (2008) Origin, spread and demography
of the Mycobacterium tuberculosis complex. PLoS Pathog., 4,
e1000160.
14. Comas,I., Homolka,S., Niemann,S. and Gagneux,S. (2009)
Genotyping of genetically monomorphic bacteria: DNA
sequencing in Mycobacterium tuberculosis highlights the
limitations of current methodologies. PLoS ONE, 4, e7815.
15. Le Fle`che,P., Fabre,M., Denoeud,F., Koeck,J. and Vergnaud,G.
(2002) High resolution, on-line identification of strains from the
Mycobacterium tuberculosis complex based on tandem repeat
typing. BMC Microbiol., 2, 37.
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