title: Investigation of bacterial and archaeal communities: novel protocols using modern sequencing by Illumina MiSeq and traditional DGGE-cloning.
abstract: Different protocols based on Illumina high-throughput DNA sequencing and denaturing gradient gel electrophoresis (DGGE)-cloning were developed and applied for investigating hot spring related samples. The study was focused on three target genes: archaeal and bacterial 16S rRNA and mcrA of methanogenic microflora. Shorter read lengths of the currently most popular technology of sequencing by Illumina do not allow analysis of the complete 16S rRNA region, or of longer gene fragments, as was the case of Sanger sequencing. Here, we demonstrate that there is no need for special indexed or tailed primer sets dedicated to short variable regions of 16S rRNA since the presented approach allows the analysis of complete bacterial 16S rRNA amplicons (V1-V9) and longer archaeal 16S rRNA and mcrA sequences. Sample augmented with transposon is represented by a set of approximately 300 bp long fragments that can be easily sequenced by Illumina MiSeq. Furthermore, a low proportion of chimeric sequences was observed. DGGE-cloning based strategies were performed combining semi-nested PCR, DGGE and clone library construction. Comparing both investigation methods, a certain degree of complementarity was observed confirming that the DGGE-cloning approach is not obsolete. Novel protocols were created for several types of laboratories, utilizing the traditional DGGE technique or using the most modern Illumina sequencing.
authors.affiliation: Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 84551, Bratislava, Slovakia.
authors.forename: Lucia
authors.initials: L
authors.lastname: Kraková
authors.affiliation: Comenius University Science Park, Ilkovičova 8, 84104, Bratislava, Slovakia.
authors.forename: Katarína
authors.initials: K
authors.lastname: Šoltys
authors.affiliation: Department of Computer Science, Faculty of Mathematics, Physics and Informatics, Comenius University, Mlynská dolina, 84248, Bratislava, Slovakia.
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Budiš
authors.affiliation: Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 84551, Bratislava, Slovakia.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Grivalský
authors.affiliation: Geneton Ltd., Ilkovičova 3, 84104, Bratislava, Slovakia.
authors.forename: František
authors.initials: F
authors.lastname: Ďuriš
authors.affiliation: Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 84551, Bratislava, Slovakia. domenico.pangallo@savba.sk.
authors.forename: Domenico
authors.initials: D
authors.lastname: Pangallo
authors.affiliation: Geneton Ltd., Ilkovičova 3, 84104, Bratislava, Slovakia.|Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Ilkovičova 6, Mlynská dolina, 84215, Bratislava, Slovakia.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Szemes
mesh_terms: D019641:DNA, Archaeal / Q000737:chemistry* / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D058645:Denaturing Gradient Gel Electrophoresis / Q000379:methods; D045482:Hot Springs / Q000382:microbiology*; D064307:Microbiota; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D019641:DNA, Archaeal; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: 16S rRNA; DGGE-cloning; Emirge; Illumina; Microbial diversity characterization; Mothur; mcrA