Comparison of Methods for Picking the Operational Taxonomic Units From Amplicon Sequences.
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ABSTRACT:
SUBMITTER: Wei ZG
PROVIDER: S-EPMC8024490 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:A variety of methods are available to collapse 16S rRNA gene sequencing reads to the operational taxonomic units (OTUs) used in microbiome analyses. A number of studies have aimed to compare the quality of the resulting OTUs. However, in the absence of a standard method to define and enumerate the different taxa within a microbial community, existing comparisons have been unable to compare the ability of clustering methods to generate units that accurately represent functional taxonomic segregation. We have previously demonstrated heritability of the microbiome and we propose this as a measure of each methods' ability to generate OTUs representing biologically relevant units. Our approach assumes that OTUs that best represent the functional units interacting with the hosts' properties will produce the highest heritability estimates. Using 1,750 unselected individuals from the TwinsUK cohort, we compared 11 approaches to OTU clustering in heritability analyses. We find that de novo clustering methods produce more heritable OTUs than reference based approaches, with VSEARCH and SUMACLUST performing well. We also show that differences resulting from each clustering method are minimal once reads are collapsed by taxonomic assignment, although sample diversity estimates are clearly influenced by OTU clustering approach. These results should help the selection of sequence clustering methods in future microbiome studies, particularly for studies of human host-microbiome interactions.
| S-EPMC5012273 | biostudies-literature
Project description:Background. 16S rRNA gene sequences are routinely assigned to operational taxonomic units (OTUs) that are then used to analyze complex microbial communities. A number of methods have been employed to carry out the assignment of 16S rRNA gene sequences to OTUs leading to confusion over which method is optimal. A recent study suggested that a clustering method should be selected based on its ability to generate stable OTU assignments that do not change as additional sequences are added to the dataset. In contrast, we contend that the quality of the OTU assignments, the ability of the method to properly represent the distances between the sequences, is more important. Methods. Our analysis implemented six de novo clustering algorithms including the single linkage, complete linkage, average linkage, abundance-based greedy clustering, distance-based greedy clustering, and Swarm and the open and closed-reference methods. Using two previously published datasets we used the Matthew's Correlation Coefficient (MCC) to assess the stability and quality of OTU assignments. Results. The stability of OTU assignments did not reflect the quality of the assignments. Depending on the dataset being analyzed, the average linkage and the distance and abundance-based greedy clustering methods generated OTUs that were more likely to represent the actual distances between sequences than the open and closed-reference methods. We also demonstrated that for the greedy algorithms VSEARCH produced assignments that were comparable to those produced by USEARCH making VSEARCH a viable free and open source alternative to USEARCH. Further interrogation of the reference-based methods indicated that when USEARCH or VSEARCH were used to identify the closest reference, the OTU assignments were sensitive to the order of the reference sequences because the reference sequences can be identical over the region being considered. More troubling was the observation that while both USEARCH and VSEARCH have a high level of sensitivity to detect reference sequences, the specificity of those matches was poor relative to the true best match. Discussion. Our analysis calls into question the quality and stability of OTU assignments generated by the open and closed-reference methods as implemented in current version of QIIME. This study demonstrates that de novo methods are the optimal method of assigning sequences into OTUs and that the quality of these assignments needs to be assessed for multiple methods to identify the optimal clustering method for a particular dataset.
| S-EPMC4675110 | biostudies-literature
Project description:Assignment of 16S rRNA gene sequences to operational taxonomic units (OTUs) is a computational bottleneck in the process of analyzing microbial communities. Although this has been an active area of research, it has been difficult to overcome the time and memory demands while improving the quality of the OTU assignments. Here, we developed a new OTU assignment algorithm that iteratively reassigns sequences to new OTUs to optimize the Matthews correlation coefficient (MCC), a measure of the quality of OTU assignments. To assess the new algorithm, OptiClust, we compared it to 10 other algorithms using 16S rRNA gene sequences from two simulated and four natural communities. Using the OptiClust algorithm, the MCC values averaged 15.2 and 16.5% higher than the OTUs generated when we used the average neighbor and distance-based greedy clustering with VSEARCH, respectively. Furthermore, on average, OptiClust was 94.6 times faster than the average neighbor algorithm and just as fast as distance-based greedy clustering with VSEARCH. An empirical analysis of the efficiency of the algorithms showed that the time and memory required to perform the algorithm scaled quadratically with the number of unique sequences in the data set. The significant improvement in the quality of the OTU assignments over previously existing methods will significantly enhance downstream analysis by limiting the splitting of similar sequences into separate OTUs and merging of dissimilar sequences into the same OTU. The development of the OptiClust algorithm represents a significant advance that is likely to have numerous other applications. IMPORTANCE The analysis of microbial communities from diverse environments using 16S rRNA gene sequencing has expanded our knowledge of the biogeography of microorganisms. An important step in this analysis is the assignment of sequences into taxonomic groups based on their similarity to sequences in a database or based on their similarity to each other, irrespective of a database. In this study, we present a new algorithm for the latter approach. The algorithm, OptiClust, seeks to optimize a metric of assignment quality by shuffling sequences between taxonomic groups. We found that OptiClust produces more robust assignments and does so in a rapid and memory-efficient manner. This advance will allow for a more robust analysis of microbial communities and the factors that shape them.
| S-EPMC5343174 | biostudies-literature
Project description:The development of high-throughput sequencing technologies has provided microbial ecologists with an efficient approach to assess bacterial diversity at an unseen depth, particularly with the recent advances in the Illumina MiSeq sequencing platform. However, analyzing such high-throughput data is posing important computational challenges, requiring specialized bioinformatics solutions at different stages during the processing pipeline, such as assembly of paired-end reads, chimera removal, correction of sequencing errors, and clustering of those sequences into Operational Taxonomic Units (OTUs). Individual algorithms grappling with each of those challenges have been combined into various bioinformatics pipelines, such as mothur, QIIME, LotuS, and USEARCH. Using a set of well-described bacterial mock communities, state-of-the-art pipelines for Illumina MiSeq amplicon sequencing data are benchmarked at the level of the amount of sequences retained, computational cost, error rate, and quality of the OTUs. In addition, a new pipeline called OCToPUS is introduced, which is making an optimal combination of different algorithms. Huge variability is observed between the different pipelines in respect to the monitored performance parameters, where in general the amount of retained reads is found to be inversely proportional to the quality of the reads. By contrast, OCToPUS achieves the lowest error rate, minimum number of spurious OTUs, and the closest correspondence to the existing community, while retaining the uppermost amount of reads when compared to other pipelines. The newly introduced pipeline translates Illumina MiSeq amplicon sequencing data into high-quality and reliable OTUs, with improved performance and accuracy compared to the currently existing pipelines.
| S-EPMC5466709 | biostudies-literature
Project description:BackgroundMicroorganisms are an important component of an aquatic ecosystem and play a critical role in the biogeochemical cycle which influences the circulation of the materials and maintains the balance in aquatic ecosystems.ObjectiveThe seasonal variation along with the impact of anthropogenic activities, water quality, bacterial community composition and dynamics in the Loktak Lake, the largest freshwater lake of North East India, located in the Indo-Burma hotspot region was assessed during post-monsoon and winter season through metagenome analysis.MethodsFive soil samples were collected during Post-monsoon and winter season from the Loktak Lake that had undergone different anthropogenic impacts. The metagenomic DNA of the soil samples was extracted using commercial metagenomic DNA extraction kits following the manufacturer's instruction. The extracted DNA was used to prepare the NGS library and sequenced in the Illumina MiSeq platform.ResultsMetagenomics analysis reveals Proteobacteria as the predominant community followed by Acidobacteria and Actinobacteria. The presence of these groups of bacteria indicates nitrogen fixation, oxidation of iron, sulfur, methane, and source of novel antibiotic candidates. The bacterial members belonging to different groups were involved in various biogeochemical processes, including fixation of carbon and nitrogen, producing streptomycin, gramicidin and perform oxidation of sulfur, sulfide, ammonia, and methane.ConclusionThe outcome of this study provides a valuable dataset representing a seasonal profile across various land use and analysis, targeting at establishing an understanding of how the microbial communities vary across the land use and the role of keystone taxa. The findings may contribute to searches for microbial bio-indicators as biodiversity markers for improving the aquatic ecosystem of the Loktak Lake.
| S-EPMC7604743 | biostudies-literature
Project description:The development of high-throughput sequencing technologies has revolutionised the field of microbial ecology via 16S rRNA gene amplicon sequencing approaches. Clustering those amplicon sequencing reads into operational taxonomic units (OTUs) using a fixed cut-off is a commonly used approach to estimate microbial diversity. A 97% threshold was chosen with the intended purpose that resulting OTUs could be interpreted as a proxy for bacterial species. Our results show that the robustness of such a generalised cut-off is questionable when applied to short amplicons only covering one or two variable regions of the 16S rRNA gene. It will lead to biases in diversity metrics and makes it hard to compare results obtained with amplicons derived with different primer sets. The method introduced within this work takes into account the differential evolutional rates of taxonomic lineages in order to define a dynamic and taxonomic-dependent OTU clustering cut-off score. For a taxonomic family consisting of species showing high evolutionary conservation in the amplified variable regions, the cut-off will be more stringent than 97%. By taking into consideration the amplified variable regions and the taxonomic family when defining this cut-off, such a threshold will lead to more robust results and closer correspondence between OTUs and species. This approach has been implemented in a publicly available software package called DynamiC.
| S-EPMC5812548 | biostudies-literature
Project description:Ninety percent of cultured bacterial nitrate reducers with a 16S rRNA gene similarity of > or =97% had a narG or nosZ similarity of > or =67% or > or =80%, respectively, suggesting that 67% and 80% could be used as standardized, conservative threshold similarity values for narG and nosZ, respectively (i.e., any two sequences that are less similar than the threshold similarity value have a very high probability of belonging to different species), for estimating species-level operational taxonomic units. Genus-level tree topologies of narG and nosZ were generally similar to those of the corresponding 16S rRNA genes. Although some genomes contained multiple copies of narG, recent horizontal gene transfer of narG was not apparent.
| S-EPMC2725509 | biostudies-literature
Project description:DNA sequence information has increasingly been used in ecological research on microbial eukaryotes. Sequence-based approaches have included studies of the total diversity of selected ecosystems, studies of the autecology of ecologically relevant species, and identification and enumeration of species of interest for human health. It is still uncommon, however, to delineate protistan species based on their genetic signatures. The reluctance to assign species-level designations based on DNA sequences is in part a consequence of the limited amount of sequence information presently available for many free-living microbial eukaryotes and in part a consequence of the problematic nature of and debate surrounding the microbial species concept. Despite the difficulties inherent in assigning species names to DNA sequences, there is a growing need to attach meaning to the burgeoning amount of sequence information entering the literature, and there is a growing desire to apply this information in ecological studies. We describe a computer-based tool that assigns DNA sequences from environmental databases to operational taxonomic units at approximately species-level distinctions. This approach provides a practical method for ecological studies of microbial eukaryotes (primarily protists) by enabling semiautomated analysis of large numbers of samples spanning great taxonomic breadth. Derivation of the algorithm was based on an analysis of complete small-subunit (18S) rRNA gene sequences and partial gene sequences obtained from the GenBank database for morphologically described protistan species. The program was tested using environmental 18S rRNA data sets for two oceanic ecosystems. A total of 388 operational taxonomic units were observed for 2,207 sequences obtained from samples collected in the western North Atlantic and eastern North Pacific oceans.
| S-EPMC2747860 | biostudies-literature
Project description:BackgroundEnterocytozoon bieneusi is a microsporidian, commonly found in animals, including humans, in various countries. However, there is scant information about this microorganism in Australasia. In the present study, we conducted the first molecular epidemiological investigation of E. bieneusi in three species of marsupials (Macropus giganteus, Vombatus ursinus and Wallabia bicolor) living in the catchment regions which supply the city of Melbourne with drinking water.MethodsGenomic DNAs were extracted from 1365 individual faecal deposits from these marsupials, including common wombat (n = 315), eastern grey kangaroo (n = 647) and swamp wallaby (n = 403) from 11 catchment areas, and then individually tested using a nested PCR-based sequencing approach employing the internal transcribed spacer (ITS) and small subunit (SSU) of nuclear ribosomal DNA as genetic markers.ResultsEnterocytozoon bieneusi was detected in 19 of the 1365 faecal samples (1.39%) from wombat (n = 1), kangaroos (n = 13) and wallabies (n = 5). The analysis of ITS sequence data revealed a known (designated NCF2) and four new (MWC_m1 to MWC_m4) genotypes of E. bieneusi. Phylogenetic analysis of ITS sequence data sets showed that MWC_m1 (from wombat) clustered with NCF2, whereas genotypes MWC_m2 (kangaroo and wallaby), MWC_m3 (wallaby) and MWC_m4 (kangaroo) formed a new, divergent clade. Phylogenetic analysis of SSU sequence data revealed that genotypes MWC_m3 and MWC_m4 formed a clade that was distinct from E. bieneusi. The genetic distinctiveness of these two genotypes suggests that they represent a new species of Enterocytozoon.ConclusionsFurther investigations of Enterocytozoon spp. from macropods and other animals will assist in clarifying the taxonomy and epidemiology of these species in Australia and elsewhere, and in assessing the public health risk of enterocytozoonosis.
| S-EPMC6022301 | biostudies-literature
Project description:The lack of a consensus bacterial species concept greatly hampers our ability to understand and organize bacterial diversity. Operational taxonomic units (OTUs), which are clustered on the basis of DNA sequence identity alone, are the most commonly used microbial diversity unit. Although it is understood that OTUs can be phylogenetically incoherent, the degree and the extent of the phylogenetic inconsistency have not been explicitly studied. Here, we tested the phylogenetic signal of OTUs in a broad range of bacterial genera from various phyla. Strikingly, we found that very few OTUs were monophyletic, and many showed evidence of multiple independent origins. Using previously established bacterial habitats as benchmarks, we showed that OTUs frequently spanned multiple ecological habitats. We demonstrated that ecological heterogeneity within OTUs is caused by their phylogenetic inconsistency, and not merely due to 'lumping' of taxa resulting from using relaxed identity cut-offs. We argue that ecotypes, as described by the Stable Ecotype Model, are phylogenetically and ecologically more consistent than OTUs and therefore could serve as an alternative unit for bacterial diversity studies. In addition, we introduce QuickES, a new wrapper program for the Ecotype Simulation algorithm, which is capable of demarcating ecotypes in data sets with tens of thousands of sequences.
| S-EPMC3664822 | biostudies-other