Lucia Krakova, Katarina Soltys, Jaroslav Budis, Tomas Grivalsky, Frantisek Duris, Domenico Pangallo, Tomas Szemes. Investigation of bacterial and archaeal communities: novel protocols using modernsequencing by Illumina MiSeq and traditional DGGE-cloning. Extremophiles, 20(5):795-808. 2016.

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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.