Appendix S1. Experimental Procedures
Fungal isolates and DNA extraction
The fungal isolates were retrieved from primary cultures initiated from a single selected
colony, having previously been identified on the basis of macroscopic and microscopic
morphological characteristics and standard mycological procedures. Subsequently,
identification was confirmed by sequencing analyses of β-tubulin and rodlet A genes
and microsatellite typing (Serrano et al., 2011; Araujo et al., 2012). Before DNA
extraction, the fungi were grown for five days on Sabouraud dextrose agar slants
(Difco) at 30 ºC. DNA (50-250 ng) was extracted from conidia using the sodium
hydroxide method (http://www.aspergillus.org.uk/indexhome.htm?secure/laboratory_
protocols) and it was suspended in 50 µL of sterile water and stored at -20 ºC.
DNA amplification and sequencing
Previously described primers for MLST amplification (Caramalho et al., 2013) and
microsatellite-multiplex (Araujo et al., 2009) were employed and the remaining primers
designed using Primer3 v.0.4.0 software (http://bioinfo.ut.ee/primer3-0.4.0/). Primerdimer
and
hairpin
structures
were
avoided
by
using
AutoDimer
v.1.0
(http://www.cstl.nist.gov/biotech/strbase/AutoDimerHomepage/AutoDimerProgramHo
mepage.htm). The list of primers is available on supplementary Table S1.
The primers were initially tested in singleplex reactions for confirmation of its
specificity. Then, multiplex PCR was performed in a total reaction volume of 5 µL, as
previously described (Caramalho et al., 2013). The amplification products were
confirmed on polyacrylamide gel and visualised through standard silver-staining (Qu et
al., 2005). Multiplex PCR products were purified with ExoSAP-IT (USB Corporation)
1
and sequencing reactions performed as previously described (Araujo et al., 2009).
Afterwards the reactions were analysed employing Sequencing 5.2 analysis software
(Applied Biosystems).
Mini-sequencing SNaPshot assays
Single base extension (SBE) primers were designed using the previously referred
AutoDimer v.1.0 software (see supplementary information Figure S1). Two minisequencing reactions were set up using 1.5 µL of purified multiplex PCR product, 1.0
µL of SBE primers mix, 1.0 µL of SNaPshot® Multiplex Reaction Ready Mix (Applied
Biosystems) and 1.5 µL of ultrapure water. The cycling conditions were 25 cycles at 96
ºC for 10 s, 50 ºC for 5 s, and 60 ºC for 30 s (Eusebio et al., 2013). Unincorporated
ddNTPs were eliminated with SAP (USB) and purified products were mixed with 9.0
µL of HiDiTM formamide (Applied Biosystems) and GeneScan-120 LIZ size standard.
Capillary electrophoresis was performed in ABI 3130xl Genetic Analyser (Applied
Biosystems) and the results of mini-sequencing analysed with GeneMapper® v.4.0
(Applied Biosystems).
Linkage disequilibrium analyses
The index of association (Ia) and rBarD values were calculated using Multilocus 1.3b
available at http://www.agapow.net/software/multilocus/ (Agapow and Burt, 2001). Ia
gives information of linkage disequilibrium allowing the comparison of individuals that
share the same allele at one locus and evaluating the possibility of sharing the same
allele at other loci. Ia is equal to zero if there is no linkage disequilibrium and this value
increases when strong linkage disequilibrium is found. The null hypothesis of complete
2
panmixia (Ia = 0) was tested with Multilocus 1.3b, by comparing the observed data set
after 1,000 randomizations.
References
Agapow, P.M., and Burt, A. (2001) Indices of multilocus linkage disequilibrium. Mol
Ecol Notes 1: 101–102.
Araujo, R., Pina-Vaz, C., Gonçalves Rodrigues, A., Amorim, A., and Gusmão, L.
(2009) Simple and highly discriminatory microsatellite-based multiplex PCR for
Aspergillus fumigatus strain typing. Clin Microbiol Infect 15: 260-266.
Araujo, R., Amorim, A., and Gusmão, L. (2012) Diversity and specificity of
microsatellites within Aspergillus section Fumigati. BMC Microbiol 12: 154.
Caramalho, R., Gusmão, L., Lackner, M., Amorim, A., and Araujo, R. (2013) SNaPAfu:
A novel single nucleotide polymorphism multiplex assay for Aspergillus fumigatus
direct detection, identification and genotyping in clinical specimens. PLoS One 8:
e75968.
Eusebio, N., Pinheiro, T., Amorim, A., Gamboa, F., Saraiva, L, Gusmão L., et al.
(2013) SNaPaer: a practical single nucleotide polymorphism multiplex assay for
genotyping of Pseudomonas aeruginosa. PLoS One 8: e66083.
Qu, L., Li, X., Wu, G., and Yang, N. (2005) Efficient and sensitive method of DNA
silver staining in polyacrylamide gels. Electrophoresis 26: 99-101.
Serrano, R., Gusmao, L., Amorim, A., and Araujo, R. (2011) Rapid identification of
Aspergillus fumigatus within the section Fumigati. BMC Microbiol 11: 82.
3