Abstract
In the past few years, many studies investigated the anaerobic digestion microbiome by means of 16S rRNA amplicon sequencing. Results obtained from these studies were compared to each other without taking into consideration the followed procedure for amplicons preparation and data analysis. This negligence was mainly due to the lack of knowledge regarding the biases influencing specific steps of the microbiome investigation process. In the present study, the main technical aspects of the 16S rRNA analysis were checked giving special attention to the approach used for high throughput sequencing. More specifically, the microbial compositions of three laboratory scale biogas reactors were analyzed before and after addition of sodium oleate by sequencing the microbiome with three different approaches: 16S rRNA amplicon sequencing, shotgun DNA and shotgun RNA. This comparative analysis revealed that, in amplicon sequencing, abundance of some taxa (Euryarchaeota and Spirochaetes) was biased by the inefficiency of universal primers to hybridize all the templates. Reliability of the results obtained was also influenced by the number of hypervariable regions under investigation. Finally, amplicon sequencing and shotgun DNA underestimated the Methanoculleus genus, probably due to the low 16S rRNA gene copy number encoded in this taxon.
Highlights
A fundamental step to uncover microbial community structure and dynamics is the taxonomic and phylogenetic classification of DNA sequences
In order to identify strengths and limitations associated with different approaches, sequences were generated using as template 16S rRNA amplicons, genomic DNA and total RNA collected from the same samples
Despite addition of long chain fatty acids has a relevant effect on microbial composition[36,37,38,39], the present study does not focused on the interpretation of biological data, but on investigation of potential biases determined by different high-throughput sequencing approaches on taxonomic results obtained
Summary
A fundamental step to uncover microbial community structure and dynamics is the taxonomic and phylogenetic classification of DNA sequences. Four fundamental factors contributed to the success of these approaches: (1) PCR amplification which allows the generation of amplicons from small amount of starting material[5], (2) high throughput sequencing and molecular barcoding supports parallel analysis of numerous samples, (3) availability of simple bioinformatics tools which simplify the analyses[6,7] and (4) the growing size of 16S rRNA gene databases[8,9] Due to these technical advances, in the last few years the number of scientific publications based on 16S rRNA amplicon sequencing underwent an impressive increase. This study led to the identification of biases associated with the use of universal primers in the PCR amplification step
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