Investigation of bacterial and archaeal communities: novel protocols using modern sequencing by Illumina MiSeq and traditional DGGE-cloning
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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.
KeywordsMicrobial diversity characterization Illumina Mothur Emirge 16S rRNA mcrA DGGE-cloning
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”.
- Lane DJ (1991) 16S/23S rRNA sequencing. Nucleic acid techniques in bacterial systematics. In: Stackenbrandt E, Goodfellow M (eds). Wiley, New York, 115–148Google Scholar
- Pires AC, Cleary DF, Almeida A, Cunha A, Dealtry S, Mendonça-Hagler LC, Smalla K, Gomes NC (2012) Denaturing gradient gel electrophoresis and barcoded pyrosequencing reveal unprecedented archaeal diversity in mangrove sediment and rhizosphere samples. Appl Environ Microbiol 78:5520–5528CrossRefPubMedPubMedCentralGoogle Scholar
- Šaková N, Sádecká J, Lejková J, Puškárová A, Koreňová J, Kolek E, Valík Ľ, Kuchta T, Pangallo D (2015) Characterization of May bryndza cheese from various regions in Slovakia based on microbiological, molecular and principal volatile odorants examination. J Food Nutr Res 54:239–251Google Scholar
- Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541CrossRefPubMedPubMedCentralGoogle Scholar
- Shi Y, TaPa M, Li C, Yang H, Zhang T, Gao Y, Sun J, Zeng J, Lin Q, Cao Z, OuTi K (2015) Diversity and space–time dynamics of endophytic archaea from sugar beet in the north slope of Tianshan Mountain revealed by 454 pyrosequencing and T-RFLP. World J Microbiol Biotechnol 31:1031–1039CrossRefPubMedGoogle Scholar
- Starke IC, Vahjen W, Pieper R, Zentek J (2014) The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons. Mol Biol Int 10 (Article ID 548683) Google Scholar