Influence of 16S rRNA target region on the outcome of microbiome studies in soil and saliva samples

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. Over the last decade, there has been a rapid rise in the development of microbiome studies because of the understanding of microbial communities as one of the main factors involved in environmental processes and in human health and disease. The affordability, reliability and high-throughput yield of data provided by next-generation sequencing (NGS) technologies has led to the substitution of classical approaches, such as culture-based methods, in the characterization of microorganisms 1. Depending on the objective of the research, amplicon sequencing or whole metagenome sequencing can be used to gain insight into microbial structural and functional patterns. Sequencing analysis of 16S rRNA subunit is the most widely used and cost effective strategy to discern the structure of the microbiome in all sorts of environments 2,3. 16S rRNA is a highly conserved and universal gene, whose sequence is about 1,500 bp long and consists of nine hypervariable regions (V1-V9) separated by conserved segments, working the latter as target sequences for primer design 4. 16S rRNA sequence has been fully characterized in a great number of bacterial species and strains, and a wide variety of primers have been designed to offer a broad applicability in the analysis of microbiome structure 2,5 , especially through the use of NGS platforms such as Illumina Miseq 3. Since Illumina Miseq does not allow the full sequencing of 16S rRNA (up to 600 bp), its hypervariable regions can be used individually or can be combined to assess the structure of bacterial communities 6. It has been shown that both the use of different primers and regions affects the outcome of microbiome studies 5,7-11 , and thus, such factors should be taken into consideration when designing a sequencing analysis. There are a considerable number of studies analyzing the effect of primers choice in the profiling of microbiome communities. It is known that the analysis of the human...

General Unified Microbiome Profiling Pipeline (GUMPP) for Large Scale, Streamlined and Reproducible Analysis of Bacterial 16S rRNA Data to Predicted Microbial Metagenomes, Enzymatic Reactions and Metabolic Pathways

“…On the other hand, single nucleotide polymorphism present within e.g., 10 copies of 16S rRNA operon within one organism represent distinct ASVs. In comparison to genus level analysis 16S rRNA variants of one organism are split to several ASV categories inflating ASV estimates of microbial taxonomic diversity and of functional diversity of underlying metagenomes [ 38 , 39 , 40 ]; (iii) In contrast, almost identical 16S rRNA copies and hence the lack of differences found within some genera do not enable stratification of species and strains present within, falsely deflating the number of present ASVs [ 10 , 38 , 39 , 40 , 41 , 42 ]; (iv) different hypervariable regions of 16S rRNA utilized in amplicon sequencing can result in additional distortion of signal relative to each other [ 43 ] hence compromising direct comparison of the results between studies utilizing distinct primers.…”

Simple amplicon sequencing library preparation for plant root microbial community profiling

“…Previous studies regarding plant microbiomes have commonly used column-based methods for DNA extraction, including bead-beating to lyse bacterial cells in plant tissues [1,10,12,[16][17][18][30][31][32][33][34][35][36][37] and soils [38][39][40][41][42][43]. The column-based method requires laborious and time-consuming procedures, as well as a large amount of sample (> 100 mg).…”

“…1). Although single-step PCR for library preparation has been previously used for plant microbiome studies [1,10,[30][31][32][33][34][35]37,38,40,43], two-step amplification avoids potential bias due to the use of different indices in each primary amplification [39]. The standard method, including the method provided by Illumina, used AMPure XP beads for PCR purification before the second PCR, and a custom protocol used for phyllosphere microbiome study used exonuclease treatment [19]; however, no study compared these different methods (which was not certified by peer review) is the author/funder.…”

Rapid Electrochemical-Based PCR-Less Microbial Quantification and Antimicrobial Susceptibility Profiling Directly From Blood and Urine With Unknown Microbial Load or Species

“…The interfering matrix components of each specimen type are removed and replaced by culture media, followed by a short 2-h antibiotic exposure using dried antibiotic stripwells consisting of different antibiotic conditions and an electrochemical sensor assay to quantify the 16S ribosomal RNA (rRNA) present in each antibiotic condition after exposure. The use of 16S rRNA as an analyte has become a common practice in many applications, such as pathogen identification, and therefore, has led to the design of a wide variety of probe pairs that detect the many characterized 16S rRNA sequences of various bacterial strains ( Soriano-Lerma et al, 2020 ). Oligonucleotide probes complementary to target 16S rRNA sequences have been used as a tool to identify bacteria at the species level and assist in differentiating between closely related bacterial species.…”