Dataset Information

Reliability of species detection in 16S microbiome analysis: Comparison of five widely used pipelines and recommendations for a more standardized approach.

ABSTRACT: The use of NGS-based testing of the bacterial microbiota is often impeded by inconsistent or non-reproducible results, especially when applying different analysis pipelines and reference databases. We investigated five frequently used software packages by submitting the same monobacterial datasets to them, representing the V1-2 and the V3-4 regions of the 16S-rRNA gene of 26 well characterized strains, which were sequenced by the Ion Torrent™ GeneStudio S5 system. The results obtained were divergent and calculations of relative abundance did not yield the expected 100%. We investigated these inconsistencies and were able to attribute them to failures either of the pipelines themselves or of the reference databases they rely on. On the basis of these findings, we recommend certain standards which should help to render microbiome testing more consistent and reproducible, and thus useful in clinical practice.

SUBMITTER: Hiergeist A

PROVIDER: S-EPMC9934417 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Reliability of species detection in 16S microbiome analysis: Comparison of five widely used pipelines and recommendations for a more standardized approach.

Hiergeist Andreas A Ruelle Jean J Emler Stefan S Gessner André A

PloS one 20230216 2

The use of NGS-based testing of the bacterial microbiota is often impeded by inconsistent or non-reproducible results, especially when applying different analysis pipelines and reference databases. We investigated five frequently used software packages by submitting the same monobacterial datasets to them, representing the V1-2 and the V3-4 regions of the 16S-rRNA gene of 26 well characterized strains, which were sequenced by the Ion Torrent™ GeneStudio S5 system. The results obtained were diver ...[more]

PMID: 36795699

Similar Datasets

Project description:BackgroundAmong older adults, decreased handgrip strength is associated with greater risk of frailty, and loss of physical function, mobility, lean mass, and overall muscular strength and power. Frailty is also associated with sarcopenia, for which handgrip strength measurement has been recommended for diagnostic purposes. Specific cutoff points for diagnosis have been identified, but use of different devices may affect measurement. Therefore to assess validity and reliability, we compared the two most frequently used devices, the Jamar hydraulic and Smedley spring handgrip dynamometers.MethodsSixty-seven older (76.2 ± 0.9 years) men (n = 34) and women (n = 33) completed two trials of handgrip strength measurement on sequential days (T1, T2) using both devices in random order. Intraclass correlations were used to assess test-retest reliability, and Bland-Altman analysis was used to assess validity as the level of agreement between devices.ResultsThere were significant (p < 0.001) relationships between devices at T1 (r = 0.94) and T2 (r = 0.94) and strong (p < 0.001) intraclass correlations were observed for both devices (Jamar = 0.98; Smedley = 0.96), indicating excellent reliability. However, there were significant differences between devices. Strength measured with Jamar was greater than Smedley at both T1 (27.4 ± 1.4 vs. 23.4 ± 1.1 kg, p < 0.001) and T2 (25.3 ± 1.4 vs. 21.8 ± 1.2 kg, p < 0.001). Bland-Altman analysis confirmed these differences. Subgroup analysis to evaluate the effect of gender and age indicated that in women and old-old (>75 years) participants, differences between devices were closer to zero for both measurements compared to men and young-old (65-75 years) participants.ConclusionsOur results demonstrate that despite excellent reliability, there is poor agreement between devices, indicating a lack of validity. For use as a diagnostic tool, standardization and device-specific cutoff points for handgrip dynamometry are needed.

Project description:Short-amplicon 16S rRNA gene sequencing is currently the method of choice for studies investigating microbiomes. However, comparative studies on differences in procedures are scarce. We sequenced human stool samples and mock communities with increasing complexity using a variety of commonly used protocols. Short amplicons targeting different variable regions (V-regions) or ranges thereof (V1-V2, V1-V3, V3-V4, V4, V4-V5, V6-V8, and V7-V9) were investigated for differences in the composition outcome due to primer choices. Next, the influence of clustering (operational taxonomic units [OTUs], zero-radius OTUs [zOTUs], and amplicon sequence variants [ASVs]), different databases (GreenGenes, the Ribosomal Database Project, Silva, the genomic-based 16S rRNA Database, and The All-Species Living Tree), and bioinformatic settings on taxonomic assignment were also investigated. We present a systematic comparison across all typically used V-regions using well-established primers. While it is known that the primer choice has a significant influence on the resulting microbial composition, we show that microbial profiles generated using different primer pairs need independent validation of performance. Further, comparing data sets across V-regions using different databases might be misleading due to differences in nomenclature (e.g., Enterorhabdus versus Adlercreutzia) and varying precisions in classification down to genus level. Overall, specific but important taxa are not picked up by certain primer pairs (e.g., Bacteroidetes is missed using primers 515F-944R) or due to the database used (e.g., Acetatifactor in GreenGenes and the genomic-based 16S rRNA Database). We found that appropriate truncation of amplicons is essential and different truncated-length combinations should be tested for each study. Finally, specific mock communities of sufficient and adequate complexity are highly recommended.IMPORTANCE In 16S rRNA gene sequencing, certain bacterial genera were found to be underrepresented or even missing in taxonomic profiles when using unsuitable primer combinations, outdated reference databases, or inadequate pipeline settings. Concerning the last, quality thresholds as well as bioinformatic settings (i.e., clustering approach, analysis pipeline, and specific adjustments such as truncation) are responsible for a number of observed differences between studies. Conclusions drawn by comparing one data set to another (e.g., between publications) appear to be problematic and require independent cross-validation using matching V-regions and uniform data processing. Therefore, we highlight the importance of a thought-out study design including sufficiently complex mock standards and appropriate V-region choice for the sample of interest. The use of processing pipelines and parameters must be tested beforehand.

Project description:BackgroundThe 16S mitochondrial rRNA gene is the most widely sequenced molecular marker in amphibian systematic studies, making it comparable to the universal CO1 barcode that is more commonly used in other animal groups. However, studies employ different primer combinations that target different lengths/regions of the 16S gene ranging from complete gene sequences (~ 1500 bp) to short fragments (~ 500 bp), the latter of which is the most ubiquitously used. Sequences of different lengths are often concatenated, compared, and/or jointly analyzed to infer phylogenetic relationships, estimate genetic divergence (p-distances), and justify the recognition of new species (species delimitation), making the 16S gene region, by far, the most influential molecular marker in amphibian systematics. Despite their ubiquitous and multifarious use, no studies have ever been conducted to evaluate the congruence and performance among the different fragment lengths.ResultsUsing empirical data derived from both Sanger-based and genomic approaches, we show that full-length 16S sequences recover the most accurate phylogenetic relationships, highest branch support, lowest variation in genetic distances (pairwise p-distances), and best-scoring species delimitation partitions. In contrast, widely used short fragments produce inaccurate phylogenetic reconstructions, lower and more variable branch support, erratic genetic distances, and low-scoring species delimitation partitions, the numbers of which are vastly overestimated. The relatively poor performance of short 16S fragments is likely due to insufficient phylogenetic information content.ConclusionsTaken together, our results demonstrate that short 16S fragments are unable to match the efficacy achieved by full-length sequences in terms of topological accuracy, heuristic branch support, genetic divergences, and species delimitation partitions, and thus, phylogenetic and taxonomic inferences that are predicated on short 16S fragments should be interpreted with caution. However, short 16S fragments can still be useful for species identification, rapid assessments, or definitively coupling complex life stages in natural history studies and faunal inventories. While the full 16S sequence performs best, it requires the use of several primer pairs that increases cost, time, and effort. As a compromise, our results demonstrate that practitioners should utilize medium-length primers in favor of the short-fragment primers because they have the potential to markedly improve phylogenetic inference and species delimitation without additional cost.

Dataset Information

Reliability of species detection in 16S microbiome analysis: Comparison of five widely used pipelines and recommendations for a more standardized approach.

Publications

Reliability of species detection in 16S microbiome analysis: Comparison of five widely used pipelines and recommendations for a more standardized approach.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets