Abstract
Polymerase chain reaction (PCR) assays targeting 16S rRNA genes followed by DNA sequencing are still important tools to characterize microbial communities present in environmental samples. However, despite the crescent number of deposited archaeal DNA sequences in databases, until now we do not have a clear picture of the effectiveness and specificity of the universal primers widely used to describe archaeal communities from different natural habitats. Therefore, in this study, we compared the phylogenetic profile obtained when Cerrado lake sediment DNA samples were submitted to 16S rDNA PCR employing three Archaea-specific primer sets commonly used. Our findings reveal that specificity of primers differed depending on the source of the analyzed DNA. Furthermore, archaeal communities revealed by each primer pair varied greatly, indicating that 16S rRNA gene primer choice affects the community profile obtained, with differences in both taxon detection and operational taxonomic unit (OTU) estimates.
Highlights
It has been extensively documented that the vast majority of existing microbial diversity consists of yet uncultured organisms (Rappé and Giovannoni, 2003; Lloyd et al, 2018)
Several methods have been employed throughout the years, such as denaturing gradient gel electrophoresis (DGGE), restriction fragment length polymorphism (RFLP), clone libraries of polymerase chain reaction (PCR) amplicons, quantitative PCR and, more recently, metagenome assembly using generation sequencing (Streit and Schmitz, 2004; GarridoCardenas and Manzano-Agugliaro, 2017)
Originally designed to describe methanogens (Großkopf et al, 1998), have been extensively used to describe archaeal communities (Nishizawa et al, 2008; Jeyanathan et al, 2011; Carnevali et al, 2018), previous results of our group revealed that they are effective in the amplification of bacterial DNA sequences, especially when these organisms are present in higher abundance in environmental samples
Summary
It has been extensively documented that the vast majority of existing microbial diversity consists of yet uncultured organisms (Rappé and Giovannoni, 2003; Lloyd et al, 2018) To access this diversity, molecular biology techniques have become a valuable asset to explore microbial communities in environmental samples. Among the methods most frequently employed in environmental studies, PCR assays targeting conserved genes have played pivotal roles both in pioneer and. Reports using both clone libraries and generation sequencing of PCR amplicons are still frequently published (Tupinambá et al, 2016; Antranikian et al, 2017; Wu et al, 2017; Belmok et al, 2019). It is widely acknowledged that this approach is not devoid of potential biases, with steps such as DNA extraction, inhibition of, or unspecific DNA amplification, generation of PCR artefacts and differential amplification all playing a crucial role in result analyses (Delmont et al, 2013)
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