INTRODUCTION {#s0}
============

The presence of methicillin-resistant *Staphylococcus aureus* (MRSA) imposes a significant burden on the public health care system, where accurate molecular typing is essential for infection control and surveillance of MRSA. The current standard MRSA nomenclature includes identification of the chromosomal background, annotated by the multilocus sequence type (ST) or clonal complex (CC) and the type of staphylococcal cassette chromosome *mec* (SCC*mec*) element (indicated by roman numerals I to XIII), sometimes with the addition of a letter indicating the SCC*mec* subtype (e.g., ST80-IVc) ([@B1]).

The key components of SCC*mec* are the *mec* and *ccr* gene complexes, which contain genes responsible for methicillin resistance and the mobility of the SCC*mec* element, respectively ([@B2]). These two gene complexes are surrounded by three highly variable joining regions (J1 to J3), which contain nonessential components of the SCC*mec* element but may harbor additional resistance genes ([@B3]). The currently known SCC*mec* elements of *S. aureus* all integrate at the same unique site in an open reading frame called *orfX*, and all have the same backbone structure with the general organization *orfX*-J3-*mec*-J2*-ccr*-J1, with the exception of SCC*mec* types VIII (4A) and IX (1C2), which have a similar organization, namely, *orfX*-J3-*ccr*-J2-*mec*-J1, as shown in [Fig. 1](#fig1){ref-type="fig"}, which was previously published and kindly allowed to be used here by Hiramatsu et al. ([@B4]). The combination of the *mec* and *ccr* gene complexes determines the SCC*mec* type, while the J1 region is used for subtyping ([@B2]). Historically, subtyping of the SCC*mec* element is only conducted for SCC*mec* types IV (2B) and V (5C2 and 5C2&5).

![Backbone structure organization of SCC*mec* elements in *S. aureus*. This figure was published previously ([@B4]) and is shown here with the kind permission of Keiichi Hiramatsu.](sph0011824720001){#fig1}

Genetic rearrangement of the SCC*mec* element can, however, result in novel elements, variants of existing SCC*mec* elements, and composite elements, hence complicating the nomenclature of SCC*mec* elements.

In 2009, the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC) established a consensus uniform nomenclature for the SCC*mec* element that defined the requirements for the annotation of new SCC*mec* elements and provided a homepage for keeping track of approved SCC*mec* elements and information about them. Currently, SCC*mec* types I to XI are listed on the homepage (<http://www.sccmec.org>), while SCC*mec* types XII (9C2) ([@B5]) and XIII (9A) have been reported and approved by the IWG-SCC but yet not listed on the homepage.

The SCC*mec* typing schemes are based on several multiplex PCR (M-PCR) protocols that do not encompass the plasticity of SCC*mec* elements, leading to an increasing proportion of nontypeable (NT) SCC*mec* elements ([@B6][@B7][@B8]). Recent advances in whole-genome sequencing (WGS) have made data essential for SCC*mec* typing available, but no bioinformatic tools for *in silico* typing of the SCC*mec* element have been available. Here, we introduce SCC*mec*Finder, an *in silico* web-based bioinformatic tool for the identification and typing of SCC*mec* elements listed by the IWG-SCC from WGS data on *S. aureus* isolates. Together with other existing tools for multilocus sequence typing (MLST) and *spa* typing, SCC*mec*Finder enables high-throughput WGS-based typing of MRSA isolates. The tool is freely available at the Center for Genomic Epidemiology (CGE) homepage, <http://genomicepidemiology.org/>.

RESULTS AND DISCUSSION {#s1}
======================

SCC*mec* typing by SCC*mec*Finder. {#s1.1}
----------------------------------

The initial evaluation with the type I to XI reference SCC*mec* elements, the composite elements SCC*mec* types IV (2B&5) and V (5C2&5), subtypes of SCC*mec* types IV (2B) and V (5C2), and the two new elements SCC*mec* types XII (9C2) and XIII (9A) revealed concordant results for all 36 SCC*mec* elements. When both SCC*mec* typing methods (SCC*mec*Finder and the M-PCR-based typing) were applied to the 93 clinical isolates ([Table 1](#tab1){ref-type="table"}), an initial concordance of 96.7% was observed ([Table 1](#tab1){ref-type="table"}). SCC*mec*Finder correctly differentiated methicillin-susceptible *S. aureus* (MSSA) isolates from MRSA isolates in all cases. Of the 50 MSSA isolates, 3 were predicted to harbor an SCC-like element having only the type 1 *ccr* gene complex by SCC*mec*Finder. M-PCR-based typing confirmed the presence of a type 1*ccr* gene complex and the absence of the *mec* gene and gene complex. All three isolates belonged to ST1/t127, which has previously been associated with the SCC-like element SCC*fus*, which contains the fusidic acid resistance gene *fusC* ([@B9]). The presence of the *fusC* gene in the three isolates was confirmed, indicating that the isolates harbor the SCC*fus* element. SCC*mec*Finder correctly predicted the SCC*mec* elements in 39 of the 43 MRSA isolates, with an average (*k*-mer) template coverage 83.83%, ranging from 44.12 to 99.79% for the 39 SCC*mec* elements.

###### 

SCC*mec* typing results and ST/*spa* type obtained by the three SCC*mec* typing methods for the MRSA strains from the clinical data set

  **Strain**   **ST/*spa* type**   **SCC*mec* typing result**   
  ------------ ------------------- ---------------------------- -----------
  75013        ST30/t019           NT                           IV (2B)
  75015        ST22/t032           IV (2B)                      IV (2B)
  75019        ST5/t002            IV (2B)                      IV (2B)
  75020        ST225/t014          NT                           II (2A)
  75024        ST152/t355          V (5C2)                      NT
  75049        ST398/t034          NT                           V (5C2&5)
  75050        ST30/t019           IV (2B)                      IV (2B)
  75052        ST87/t216           IV (2B)                      IV (2B)
  75054        ST398/t034          V (5C2)                      V (5C2&5)
  75055        ST30/t1752          IV (2B)                      IV (2B)
  75056        ST8/t008            IV (2B)                      IV (2B)
  75058        ST6/t4403           NT                           IV (2B)
  75061        ST30/t019           IV (2B)                      IV (2B)
  75083        ST22/t3638          IV (2B)                      IV (2B&5)
  75098        ST5/t1062           IV (2B)                      IV (2B)
  75100        ST8/t008            IV (2B)                      IV (2B)
  75101        ST87/t217           IV (2B)                      IV (2B)
  75103        ST30/t012           IV (2B)                      IV (2B)
  75104        ST5/t5530           I (1B)                       I (1B)
  75108        ST45/t026           IV (2B)                      IV (2B)
  75125        ST5/t002            IV (2B)                      IV (2B)
  75126        ST45/t362           IV (2B)                      IV (2B)
  75127        ST45/t362           IV (2B)                      IV (2B)
  75129        ST22/t022           IV (2B)                      IV (2B)
  75146        ST398/t034          V (5C2)                      V (5C2&5)
  75147        ST1/t127            IV (2B)                      IV (2B)
  75149        ST22/t223           IV (2B)                      NT
  75150        ST45/t026           IV (2B)                      IV (2B)
  75178        ST772/t657          V (5C2)                      NT
  75180        ST5/t002            IV (2B)                      NT
  75181        ST30/t019           IV (2B)                      IV (2B)
  75182        ST5/t002            IV (2B)                      IV (2B)
  75201        ST30/unknown        IV (2B)                      IV (2B)
  75203        ST22/t032           IV (2B)                      IV (2B)
  75205        ST5/t045            II (2A)                      II (2A)
  75208        ST30/t019           IV (2B)                      IV (2B)
  75209        ST88/t690           IV (2B)                      IV (2B)
  75211        ST22/t379           IV (2B)                      IV (2B)
  75212        ST5/t010            I (1B)                       I (1B)
  75316        ST22/t022           IV (2B&5)                    IV (2B&5)
  75319        ST8/t008            IV (2B)                      IV (2B)
  75336        ST152/t355          V (5C2)                      V (5C2&5)
  75339        ST398/t011          V (5C2)                      V (5C2&5)

The PCR-based SCC*mec* typing protocol described by Kondo et al. ([@B6]).

SCC*mec*Finder and the M-PCR-based typing gave NT results for four isolates but without overlaps. Of the four isolates NT by SCC*mec*Finder, two could be recovered by a reassembly of the raw reads using SPAdes instead of the default assembler, e.g., Velvet, and one SCC*mec* element was recovered by modifying the database to the extended database, while the last isolate could not be recovered ([Table 2](#tab2){ref-type="table"}). Upon modification of the thresholds, the percentage of concordance increased to 98.9%.

###### 

Recovery of NT SCC*mec* elements

  -----------------------------------------------------------------------------------------------------------------------------------------
  **Strain**   **ST/*spa* type**   **SCC*mec* typing result**   **Recovery method**    **Final SCC*mec* type**                  
  ------------ ------------------- ---------------------------- ---------------------- ---------------------------------------- -----------
  75024        ST152/t355          V (5C2)                      NT                     Change from Velvet to SPAdes assembler   V (5C2&5)

  75149        ST22/t223           IV (2B)                      NT                     Change from Velvet to SPAdes assembler   IV (2B)

  75178        ST772/t657          V (5C2)                      V (5C2) or V (5C2&5)   Change from reference to extended\       V (5C2)
                                                                                       database                                 

  75180        ST5/t002            IV (2B)                      IV (2B) or II (2A)                                              NT
  -----------------------------------------------------------------------------------------------------------------------------------------

The PCR-based SCC*mec* typing protocol described by Kondo et al. ([@B6]).

Publically available *S. aureus* genomes from the NCBI RefSeq database (*n* = 6,852) were included to evaluate the performance of SCC*mec*Finder on genomes with different sequencing qualities from different platforms and sources to catch possible systematic errors by SCC*mec*Finder and identify the most prevalent warning messages. SCC*mec*Finder identified 6,021 genomes as MRSA and 831 as MSSA ([Table 3](#tab3){ref-type="table"}). Less than 8% of the predictions were accompanied by a warning. The predominant warning was the detection of multiple (more than one) *ccr* gene complexes and does not count for SCC*mec* types V (5C2&5) and IV (2B&5), as these are detectable by SCC*mec*Finder. Detection of multiple gene complexes complicates the Basic Local Alignment Search Tool (BLAST)-based prediction, as the method is unable to resolve which gene complexes make up the SCC*mec* element and which may be part of an SCC-like element, and hence, in such cases, SCC*mec*Finder reports the detection of multiple gene complexes to alert the user to make a final decision. Failure of the BLAST-based approach to give a definitive SCC*mec* prediction was also among the most frequently observed warnings, often because of stringent threshold levels or insufficient assembly of contigs. SCC*mec*Finder always detected SCC*mec* types VII (5C1) and X (7C1) by only one of the approaches, which could be indicative of a systematic error by SCC*mec*Finder. However, further investigation revealed no systematic errors.

###### 

SCC*mec* typing results produced by SCC*mec*Finder for *S. aureus* genomes available from the NCBI RefSeq database

  *S. aureus* group and SCC*mec* typing result                                         No. of genomes                             
  ------------------------------------------------------------------------------------ ------------------------------------------ ------------------------------------------
  MRSA                                                                                                                            
      No alerts                                                                                                                   
          SCC*mec* type I (1B)                                                         201                                        201
          SCC*mec* type II (2A)                                                        2,241                                      2,241
          SCC*mec* type III (3A)                                                       136                                        136
          SCC*mec* type IV (2B)                                                        2,616                                      2,617
          SCC*mec* type IV (2B&5)                                                      20                                         20
          SCC*mec* type V (5C2)                                                        0                                          34
          SCC*mec* type V (5C2&5)                                                      341                                        342
          SCC*mec* type VI (4B)                                                        3                                          3
          SCC*mec* type VII (5C1)                                                      0                                          0
          SCC*mec* type VIII (4A)                                                      5                                          5
          SCC*mec* type IX (1C2)                                                       0                                          0
          SCC*mec* type X (7C1)                                                        0                                          0
          SCC*mec* type XI (8E)                                                        8                                          8
      Alerts                                                                                                                      
          Multiple complexes                                                                                                      
              *ccr* type 1, *ccr* type 2, *mec* class B                                8                                          8
              *ccr* type 1, *ccr* type 4, *ccr* type 5, *ccr* type 5, *mec* class C2   1                                          1
              *ccr* type 2, *ccr* type 4, *mec* class A                                168[^c^](#ngtab3.3){ref-type="table-fn"}   168[^c^](#ngtab3.3){ref-type="table-fn"}
              *ccr* type 2, *ccr* type 4, *mec* class B                                11                                         11
              *ccr* type 2, *ccr* type 4, *ccr* type 5, *mec* class A                  8                                          8
          Contradictory prediction at:                                                                                            
              Type level                                                               76                                         67
              Subtype level                                                            76                                         76
          Failure of BLAST                                                             73                                         73
          Failure of *k*-mer                                                           27                                         1
          SCC-like                                                                     2                                          2
  MSSA                                                                                                                            
      No alerts, no SCC*mec* element                                                   773                                        773
      Alerts                                                                                                                      
          Failure of BLAST                                                             35                                         34
          SCC-like                                                                     23                                         23

In all, 6,021 MRSA and 831 MSSA isolates were tested.

The modifications included lowering the thresholds to \>50% nucleotide identity and \>50% template coverage and changing from the reference database to the extended database. In all, 6,022 MRSA and 830 MSSA isolates were tested.

A total of 147/168 strains causing alerts belonged to the same ST (ST5), indicating a single NT element.

SCC*mec* subtype prediction by SCC*mec*Finder. {#s1.2}
----------------------------------------------

The subtyping predictions by SCC*mec*Finder of SCC*mec* types IV (2B) and V (5C2) were initially evaluated by using the reference elements listed by the IWG-SCC. The evaluation showed perfect concordance. The subtyping predictions were further evaluated by comparing the findings of SCC*mec*Finder with those of PCR-based SCC*mec* typing ([Table 4](#tab4){ref-type="table"}). Sorting on the basis of the ST type, *spa* type, and subtype predicted by SCC*mec*Finder revealed a clustering of the isolates. Comparing the clustering results with the expected subtype prediction based on the multilocus ST, the *spa* type, a literature search, and PCR-based subtyping revealed a high concordance of the subtyping predictions, indicating that the SCC*mec* (sub)types are relatively stable within the ST/*spa* type clone ([Table 4](#tab4){ref-type="table"}).

###### 

Comparison of the subtype predictions of SCC*mec* types IV (2B) and V (5C2) from the clinical data set by the three SCC*mec* typing methods

  **Strain**   **ST/*spa* type**   **SCC*mec* typing result**   
  ------------ ------------------- ---------------------------- ------------------------------------------------
  75178        ST772/t657          NT                           Va (5C2)
  75024        ST152/t355          NT                           Vnt (5C2&5)
  75336        ST152/t355          NT                           Vc (5C2&5)
  75054        ST398/t034          NT                           Vc (5C2&5)
  75339        ST398/t011          NT                           Vc (5C2&5)
  75049        ST398/t034          NT                           Vc (5C2&5)
  75013        ST30/t019           IVc (2B)                     IVc (2B)
  75050        ST30/t019           IVc (2B)                     IVc (2B)
  75055        ST30/t1752          IVc (2B)                     IVc (2B)
  75061        ST30/t019           IVc (2B)                     IVc (2B)
  75181        ST5/t002            IVc (2B)                     IVc (2B)
  75208        ST30/t019           IVc (2B)                     IVc (2B)
  75108        ST45/t026           IVa (2B)                     IVa (2B)
  75150        ST45/t026           IVa (2B)                     IVa (2B)
  75126        ST45/t362           IVb (2B)                     IVb (2B)
  75127        ST45/t362           IVb (2B)                     IVb (2B)
  75052        ST87/t216           NT                           IVg (2B)
  75101        ST87/t216           NT                           IVg (2B)
  75015        ST22/t032           NT                           IVnt (2B)[^b^](#ngtab4.2){ref-type="table-fn"}
  75129        ST22/t022           NT                           IVnt (2B)[^b^](#ngtab4.2){ref-type="table-fn"}
  75203        ST22/t032           NT                           IVnt (2B)[^b^](#ngtab4.2){ref-type="table-fn"}
  75211        ST22/t379           NT                           IVnt (2B)[^b^](#ngtab4.2){ref-type="table-fn"}
  75098        ST5/t1062           IVc (2B)                     IVc (2B)
  75180        ST5/t002            IVc (2B)                     IVc (2B)
  75019        ST5/t002            IVc (2B)                     IVc (2B)
  75125        ST5/t002            IVc (2B)                     IVc (2B)
  75182        ST5/t002            IVc (2B)                     IVc (2B)
  75201        ST30/t019           IVc (2B)                     IVc (2B)
  75103        ST30/t012           IVc (2B)                     IVc (2B)
  75319        ST8/t008            IVa (2B)                     IVa (2B)
  75056        ST8/t008            IVc (2B)                     IVc (2B)
  75100        ST8/t008            NT                           IVnt (2B)[^c^](#ngtab4.3){ref-type="table-fn"}
  75147        ST1/t127            IVa (2B)                     IVa (2B)
  75058        ST6/t4403           IVa (2B)                     IVa (2B)
  75209        ST88/t690           IVa (2B)                     IVa (2B)

The PCR-based SCC*mec* typing protocol described by Kondo et al. ([@B6]).

Subtype either IVh (2B) or IVj (2B).

Contradictory subtype prediction.

Database curation and identification of novel (sub)types. {#s1.3}
---------------------------------------------------------

SCC*mec*Finder is designed to detect all of the reference SCC*mec* types listed by the IWG-SCC, and ideally, novel SCC*mec* (sub)types will be added to the databases by the curator after agreement with the IWG-SCC group upon the detection of a novel SCC*mec* (sub)type. Likewise, we suggest that notification of the IWG-SCC should be reported in the article describing the element.

The combination of two different approaches for the prediction of the SCC*mec* element in SCC*mec*Finder creates an opportunity for the user to assess whether the SCC*mec* element in question is a variant of an existing element or a novel element, as SCC*mec*Finder provides data on the detected target genes and the *k*-mer template coverage. An example is *S. aureus* JCSC7481 (GenBank accession no. [AB774378](https://www.ncbi.nlm.nih.gov/nuccore/AB774378)), which the authors have stated is a variant of SCC*mec* type V (5C2). Running the SCC*mec* element of *S. aureus* JCSC7481 through SCC*mec*Finder by using the reference database, an alert about contradictory predictions was produced. The *k*-mer template coverage was 42.4%, indicating that the element differed from the reference elements and in concordance with the conclusion of the authors that it was a variant of SCC*mec* type V (5C2). However, the element of *S. aureus* JCSC7481 has, to our knowledge, not yet been assigned by the IWG-SCC, and hence, the SCC*mec* element has not yet been added to the reference database (but is currently found in the extended database). Of importance; researchers are encouraged to consult members of the IWG-SCC and the curator of the SCC*mec*Finder database to keep the number of NT SCC*mec* elements low.

Conclusion. {#s1.4}
-----------

In conclusion, SCC*mec*Finder (<https://cge.cbs.dtu.dk/services/SCCmecFinder>) is a validated tool for rapid whole-genome-based SCC*mec* typing of MRSA that, together with the existing tools for the *in silico* prediction of the multilocus ST (<https://cge.cbs.dtu.dk/services/MLST>) and *spa* type (<https://cge.cbs.dtu.dk/services/spatyper>), can provide a standardized nomenclature of *S. aureus* genomes. It is our intention that the SCC*mec*Finder database will be curated in a way that fulfills future needs to encompass the changing epidemiology of MRSA accompanied by increasing genetic diversity.

MATERIALS AND METHODS {#s2}
=====================

Construction of SCC*mec*Finder. {#s2.1}
-------------------------------

SCC*mec*Finder combines two existing and validated gene prediction algorithms ([@B10], [@B11]), a BLAST-based approach and a *k*-mer-based approach. By utilizing the BLAST approach (BLASTn algorithm) with default thresholds set at ≥90% nucleotide identity and a minimum length of 60%, SCC*mec*Finder detects the presence of selected target genes, identifies the *mec* and *ccr* gene complex, and types the SCC*mec* element, whereas by utilizing the *k*-mer-approach, SCC*mec*Finder types the element on the basis of homology to reference SCC*mec* elements by identifying the number of co-occurring DNA sequences with the length *k*, known as *k*-mers, between the query genome and the entries in the curated database, of which only hits with ≥50% *k*-mer template coverage are considered ([Fig. 2](#fig2){ref-type="fig"}). Less stringent thresholds for the BLAST-based approach can be set, but less stringent thresholds could lead to outputs that require more downstream analysis and might give misleading annotations. The software can be found at <https://bitbucket.org/genomicepidemiology/sccmecfinder.git>. Prior for choosing the thresholds for each algorithm, a parameterization study was conducted.

![Workflow describing the decision-making of SCC*mec*Finder. The output (c) combines interpretations from the BLAST (a)- and *k*-mer (b)-based approaches. The output can be no, one, or more than one SCC*mec* element detected.](sph0011824720002){#fig2}

SCC*mec*Finder compares the predicted SCC*mec* types from the BLAST- and the *k*-mer-based predictions and checks for contradictory results to achieve correct SCC*mec* annotation. Identification of SCC*mec* type IV (2B) or V (5C2) results in subtyping of the element. Subtyping is performed as with the typing of the SCC*mec* element, on the basis of a BLAST search for selected subtyping target genes from the J1 region and the *k*-mer-based approach. On the basis of the comparison, SCC*mec*Finder outputs one or more of the five possible output types, e.g., notification of a positive/negative prediction, a contradictory SCC*mec* type/subtype, a missing/additional gene(s), the detection of multiple gene complexes, or the presence of a possible SCC-like element. If one of the prediction approaches fails, the output will be negative but will contain information about the possible presence of an SCC*mec* element to guide the user to perform further analysis ([Fig. 2](#fig2){ref-type="fig"}).

Sequence databases for SCC*mec*Finder. {#s2.2}
--------------------------------------

Two databases were developed to support the two different approaches. For the BLAST-based approach, a collection of 37 target gene sequences ([Table S1](#tabS1){ref-type="supplementary-material"} and [S2](#tabS2){ref-type="supplementary-material"}) and for the *k*-mer-based approach, a collection of 36 archetypal reference SCC*mec* were selected to represent sequences of known types available at/according to the IWG-SCC ([Table S3](#tabS3){ref-type="supplementary-material"}). Additionally, an extended version of the database for the *k*-mer-based approach, containing all of the archetypal reference SCC*mec* elements and variants of SCC*mec* type V (5C2 and 5C2&5) from *S. aureus* that have not yet been validated or assigned a subtype by the IWG-SCC, was also generated ([Table S4](#tabS4){ref-type="supplementary-material"}). Each entry in all three databases is accompanied by an NCBI GenBank accession number, thus allowing for the correlation of an identified gene(s)/element(s) in the query genome with an annotated gene(s)/element(s) in the archetypal reference SCC*mec* elements. The databases for SCC*mec*Finder can be found at <https://bitbucket.org/genomicepidemiology/sccmecfinder_db.git>.

10.1128/mSphere.00612-17.1

Target genes included in the database and used for detection of the *mec* and *ccr* gene complexes for BLAST-based SCC*mec* typing. Download TABLE S1, PDF file, 0.1 MB.

Copyright © 2018 Kaya et al.

2018

Kaya et al.

This content is distributed under the terms of the

Creative Commons Attribution 4.0 International license

.

10.1128/mSphere.00612-17.2

Subtype target genes included in the database for the BLAST-based approach and used for SCC*mec* types IV (2B) and V (5C2 and 5C2&5) in BLAST-based SCC*mec* typing. Download TABLE S2, PDF file, 0.1 MB.

Copyright © 2018 Kaya et al.

2018

Kaya et al.

This content is distributed under the terms of the

Creative Commons Attribution 4.0 International license

.

10.1128/mSphere.00612-17.3

SCC*mec* elements included in the reference database used for the *k*-mer-based approach. Download TABLE S3, PDF file, 0.1 MB.

Copyright © 2018 Kaya et al.

2018

Kaya et al.

This content is distributed under the terms of the

Creative Commons Attribution 4.0 International license

.

10.1128/mSphere.00612-17.4

SCC*mec* elements included in the extended database used for the *k*-mer-based approach. Download TABLE S4, PDF file, 0.1 MB.

Copyright © 2018 Kaya et al.

2018

Kaya et al.

This content is distributed under the terms of the

Creative Commons Attribution 4.0 International license

.

User interface and use of SCC*mec*Finder. {#s2.3}
-----------------------------------------

SCC*mec*Finder builds on the common design of the CGE user interface. The upload page consists of four dropdown menus by which the type of sequencing platform can be chosen and default settings for the analysis can be changed. The output page consists of three sections, with the first reporting the presence/absence of the *mecA*/*mecC* gene. The second section is the actual output of the SCC*mec* prediction, in which the *mec* and *ccr* gene complexes, SCC*mec* type, and possible subtype from both approaches are reported. The third and last section is reporting of the genes detected by the BLAST-based approach and the top five hits from the *k*-mer-based approach.

Submission to SCC*mec*Finder can be done by using either preassembled draft genomes or raw reads from the 454, Ion Torrent, or Illumina sequencing platform. Prior to the BLAST-based prediction, 454 and Ion Torrent raw reads are *de novo* assembled by using Newbler, while Illumina raw reads are *de novo* assembled by using Velvet ([@B10]).

SCC*mec* typing using SCC*mec*Finder and validation. {#s2.4}
----------------------------------------------------

The integrity of SCC*mec*Finder was initially evaluated by submitting reference genomes and the archetypal SCC*mec* elements included in the *k*-mer reference database by using the default thresholds. After the initial validation process, draft genomes of 93 clinical isolates were submitted to SCC*mec*Finder without further processing.

The isolates covered a diverse collection of 43 MRSA and 50 MSSA strains representing 13 different STs of predominant MRSA and MSSA clones in Denmark. The genomes were obtained after genomic DNA extraction (DNeasy Blood and Tissue kit; Qiagen, Copenhagen, Denmark), and fragment libraries were constructed by using an in-house library protocol ([@B12]), followed by 100-bp paired-end sequencing (HiSeq; Illumina) in accordance with the manufacturer's instructions. *De novo* assembly was performed with the modified Velvet algorithm integrated into the web tool Assembler v1.2 (available at <https://cge.cbs.dtu.dk/services/Assembler/>). The *in silico* predictions by SCC*mec*Finder were evaluated by comparing the results with those predicted by the PCR-based protocol of Kondo et al. ([@B6]), M-PCRs 1 and 2. Subtyping were performed by M-PCR 3 as described by Kondo et al. ([@B6]). Composite elements were detected by ensuring the presence of both *ccr* gene complexes in SCC*mec* types IV (2B&5) and V (5C2&5), and an in-house protocol was used for the typing of *ccr*C1 gene alleles 2 and 8, respectively, for the verification of SCC*mec* type V (5C2&5) ([@B13]).

Additional genomes. {#s2.5}
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Finally, 6,852 publically available *S. aureus* draft genomes from the NCBI Reference Sequence Database (RefSeq \[<https://ncbi.nlm.nih.gov/refseq/>\], accessed 16 September 2016) were downloaded to validate the SCC*mec*Finder outputs for any systematic errors. The assemblies were subjected to rough quality filtering on the basis of being *S. aureus* (determined on the basis of MLST and single nucleotide polymorphism and phylogenetic analyses), their genome size, number of contigs, and *N*~50~ value, where genomes with a size of \<2.5 Mb, an *N*~50~ value of \<10,000, and \>1,000 contigs were discarded. All genomes were subjected to MLST (<http://github.com/tseemann/mlst>) and subsequently submitted to SCC*mec*Finder by using default thresholds.

Accession number(s). {#s2.6}
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All of clinical sequences included in this study are available at the European Nucleotide Archive under project accession number [PRJEB22380](https://www.ebi.ac.uk/ena/data/search?query=PRJEB22380).

This work was supported by the Danish Ministry of Health and Prevention as part of the surveillance of MRSA, the Danish Council for Strategic Research (grant 09-067103), and the European Union Horizon 2020 research and innovation program under grant agreement 643476 to the COMPARE project (<http://www.compare-europe.eu>).

[^1]: Present address: Hülya Kaya, Statens Serum Institut, Copenhagen, Denmark.

[^2]: **Citation** Kaya H, Hasman H, Larsen J, Stegger M, Johannesen TB, Allesøe RL, Lemvigh CK, Aarestrup FM, Lund O, Larsen AR. 2018. SCC*mec*Finder, a web-based tool for typing of staphylococcal cassette chromosome *mec* in *Staphylococcus aureus* using whole-genome sequence data. mSphere 3:e00612-17. <https://doi.org/10.1128/mSphere.00612-17>.
