Abstract
Next generation sequencing methods are widely used in evaluating the structure and functioning of microbial communities, especially those centered on 16S rRNA subunit. Since Illumina Miseq, the most used sequencing platform, does not allow the full sequencing of 16S rRNA gene, this study aims to evaluate whether the choice of different target regions might affect the outcome of microbiome studies regarding soil and saliva samples. V1V3, V3V4, V4V5 and V6V8 domains were studied, finding that while some regions showed differences in the detection of certain bacterial taxa and in the calculation of alpha diversity, especially in soil samples, the overall effect did not compromise the differentiation of any sample type in terms of taxonomic analysis at the genus level. 16S rRNA target regions did affect the detection of specific bacteria related to soil quality and development, and microbial genera used as health biomarkers in saliva. V1V3 region showed the closest similarity to internal sequencing control mock community B, suggesting it might be the most preferable choice regarding data reliability.
Highlights
Generation sequencing methods are widely used in evaluating the structure and functioning of microbial communities, especially those centered on 16S rRNA subunit
Studies addressing the detailed impact of 16S rRNA regions on the profiling of bacterial communities in both environmental and biological samples have not been published to date, and nor have comprehensive analysis including the use of combined regions such as V1V3, V3V4, V4V5 and V6V8, the most commonly used to increase taxonomic accuracy in amplicon sequencing
Sequencing of 16S rRNA gene amplicons with Illumina MiSeq resulted in a total of 5308825 sequences after bioinformatic processing (1208831 reads for V1V3, 1250262 for V3V4, 1497786 for V4V5 and 1351946 for V6–V8), with an average length of 492 bp for V1V3, 457 bp for V3V4, 412 bp for V4V5 and 438 bp for V6V8 (447 bp overall mean length)
Summary
Generation sequencing methods are widely used in evaluating the structure and functioning of microbial communities, especially those centered on 16S rRNA subunit. Since Illumina Miseq, the most used sequencing platform, does not allow the full sequencing of 16S rRNA gene, this study aims to evaluate whether the choice of different target regions might affect the outcome of microbiome studies regarding soil and saliva samples. Studies addressing the detailed impact of 16S rRNA regions on the profiling of bacterial communities in both environmental and biological samples have not been published to date, and nor have comprehensive analysis including the use of combined regions such as V1V3, V3V4, V4V5 and V6V8, the most commonly used to increase taxonomic accuracy in amplicon sequencing. Soils were selected as representatives for highly diverse environmental s amples[12] and so were saliva samples as its biological counterparts[13], since no scientific articles analyzing the effect of 16S rRNA target regions have been published regarding those samples types
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