Comparing DADA2 and OTU clustering approaches in studying the bacterial communities of atopic dermatitis

Introduction. The pathogenesis of atopic dermatitis (AD) is not yet fully understood, but the bacterial composition of AD patients’ skin has been shown to have an increased abundance of Staphylococcus aureus . More recently, coagulase-negative Staphylococcus (CoNS) species were shown to be able to inhibit S. aureus , but further studies are required to determine the effects of Staphylococcus community variation in AD. Aim. Here we investigated whether analysing metabarcoding data with the more recently developed DADA2 approach improves metabarcoding analyses compared to the previously used operational taxonomic unit (OTU) clustering, and can be used to study Staphylococcus community dynamics. Methods. The bacterial 16S rRNA region from tape strip samples of the stratum corneum of AD patients (non-lesional skin) and non-AD controls was metabarcoded. We processed metabarcoding data with two different bioinformatic pipelines (an OTU clustering method and DADA2), which were analysed with and without technical replication (sampling strategy). Results. We found that OTU clustering and DADA2 performed well for community-level studies, as demonstrated by the identification of significant differences in the skin bacterial communities associated with AD. However, the OTU clustering approach inflated bacterial richness, which was worsened by not having technical replication. Data processed with DADA2 likely handled sequencing errors more effectively and thereby did not inflate molecular richness. Conclusion. We believe that DADA2 represents an improvement over an OTU clustering approach, and that biological replication rather than technical replication is a more effective use of resources. However, neither OTU clustering nor DADA2 gave insights into Staphylococcus community dynamics, and caution should remain in not overinterpreting the taxonomic assignments at lower taxonomic ranks.

An efficient and robust laboratory workflow and tetrapod database for larger scale eDNA studies

BackgroundThe use of environmental DNA, ‘eDNA,’ for species detection via metabarcoding is growing rapidly. We present a co-designed lab workflow and bioinformatic pipeline to mitigate the two most important risks of eDNA: sample contamination and taxonomic mis-assignment. These risks arise from the need for PCR amplification to detect the trace amounts of DNA combined with the necessity of using short target regions due to DNA degradation.FindingsOur high-throughput workflow minimises these risks via a four-step strategy: (1) technical replication with two PCR replicates and two extraction replicates; (2) using multi-markers (12S, 16S, CytB); (3) a ‘twin-tagging,’ two-step PCR protocol;(4) use of the probabilistic taxonomic assignment method PROTAX, which can account for incomplete reference databases.As annotation errors in the reference sequences can result in taxonomic mis-assignment, we supply a protocol for curating sequence datasets. For some taxonomic groups and some markers, curation resulted in over 50% of sequences being deleted from public reference databases, due to (1) limited overlap between our target amplicon and reference sequences; (2) mislabelling of reference sequences; (3) redundancy.Finally, we provide a bioinformatic pipeline to process amplicons and conduct PROTAX assignment and tested it on an ‘invertebrate derived DNA’ (iDNA) dataset from 1532 leeches from Sabah, Malaysia. Twin-tagging allowed us to detect and exclude sequences with non-matching tags. The smallest DNA fragment (16S) amplified most frequently for all samples, but was less powerful for discriminating at species rank. Using a stringent and lax acceptance criteria we found 162 (stringent) and 190 (lax) vertebrate detections of 95 (stringent) and 109 (lax) leech samples.ConclusionsOur metabarcoding workflow should help research groups increase the robustness of their results and therefore facilitate wider usage of e/iDNA, which is turning into a valuable source of ecological and conservation information on tetrapods.

Not so different in present attitudes and behaviour, but expected future membership: A technical replication of a study of party youth in six European democracies

Based on the statistical analysis of an original survey of young party members from six European democracies, a study concluded that three types of young members differed systematically regarding their membership objectives, activism, efficacy and perceptions of the party and self-perceived political future. We performed a technical replication of the original study, correcting four deficiencies, which led us to a different conclusion. First, we discuss substantive significance in addition to statistical significance. Second, we ran significance tests on all comparisons instead of limiting them to an arbitrary subset. Third, we performed pairwise comparisons between the three types of members instead of using pooled groups. Fourth, we avoided the inflation of the type-I error rate due to multiple testing by using the Bonferroni–Holm correction. We found that most of the differences between the types lacked substantive significance, and that statistical significance only coherently distinguished the types of members in their future membership, but not in their present behaviour and attitudes.

Dynamic Negotiation Mechanism for Improving Service Quality for Replicas in Data Grids

In order not to be limited in term of calculation, storage and communication, the concept of grid, which does not cease evolving, makes it possible to offer a practical operation of work unified as well as a great storage and computing power. To manage the division in the data grid, technical replication is used, but in spite of their advantages, the competitor access to the data could involve inconsistencies, from where the great challenge to ensure the consistency management between replicas of object. In this chapter, we describe model double-layered adapted to the applications on a large scale and which represents the support of the hybrid approach of consistency management of replicas based on pessimistic and optimistic approaches. This hybrid approach present an adapted mechanism based on the various negotiation forms between virtual consistency agents to be able to reduce the number of conflicts between replicas in data grids.