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Investigation of bacterial and archaeal communities: novel protocols using modern sequencing by Illumina MiSeq and traditional DGGE-cloning

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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.

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Acknowledgments

This publication is the result of the projects’ implementation: Comenius University Science Park – 2.phase and REVOGENE—Research centre for molecular genetics (ITMS 26240220067) supported by the Research & Innovation Operational Programme funded by the European Regional Development Fund. This study was also supported by SAS-MOST Joint Research Cooperation (Bilateral project Slovakia-Taiwan) No. SAS-MOST JRP 2014/3: “Exploring Microbial Diversity and Functionality in Thermophilic Bioreactors for Innovation in Biotechnology”.

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Authors and Affiliations

  1. Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 84551, Bratislava, Slovakia

    Lucia Kraková, Tomáš Grivalský & Domenico Pangallo

  2. Comenius University Science Park, Ilkovičova 8, 84104, Bratislava, Slovakia

    Katarína Šoltys

  3. Department of Computer Science, Faculty of Mathematics, Physics and Informatics, Comenius University, Mlynská dolina, 84248, Bratislava, Slovakia

    Jaroslav Budiš

  4. Geneton Ltd., Ilkovičova 3, 84104, Bratislava, Slovakia

    František Ďuriš & Tomáš Szemes

  5. Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Ilkovičova 6, Mlynská dolina, 84215, Bratislava, Slovakia

    Tomáš Szemes

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  1. Lucia Kraková
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  2. Katarína Šoltys
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  3. Jaroslav Budiš
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  4. Tomáš Grivalský
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  5. František Ďuriš
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  6. Domenico Pangallo
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  7. Tomáš Szemes
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Correspondence to Domenico Pangallo.

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Communicated by A. Oren.

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792_2016_855_MOESM1_ESM.pdf

Fig. S1. Krona graphs of microorganisms content in sediment and water samples using 16S rRNA universal archaeal primers and analyzed by DGGE-cloning and next generation sequencing (by Mothur and Emirge bioinformatics tools). Fig. S2. Krona graphs of microorganisms content in sediment and water samples using specific primers for archaeal mcrA gene and analyzed by DGGE-cloning and next generation sequencing (by Mothur and Emirge bioinformatics tools). Table S1. Diversity and percentage of bacterial clones detected by semi-nested PCR-DGGE and clone library strategy oriented to 16S rRNA gene. Table S2. Diversity and percentage of archaeal clones detected by semi-nested PCR-DGGE and clone library strategy oriented to 16S rRNA gene. Table S3. Diversity and percentage of methanogen clones detected by semi-nested PCR-DGGE and clone library strategy oriented to mcrA gene. (PDF 537 kb)

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Kraková, L., Šoltys, K., Budiš, J. et al. Investigation of bacterial and archaeal communities: novel protocols using modern sequencing by Illumina MiSeq and traditional DGGE-cloning. Extremophiles 20, 795–808 (2016). https://doi.org/10.1007/s00792-016-0855-5

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