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

Abstract

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.

Temporal and Spatial Variation of the Skin-Associated Bacteria from Healthy Participants and Atopic Dermatitis Patients

“…At each sampling time point, the stratum corneum was collected using a tape-stripping technique ( 22 , 39 ). Five consecutive adhesive, d -Squame tapes (22 mm diameter; CuDerm, Dallas TX, USA) were placed onto the skin and pressed for 10 s with standardized pressure (225 g/cm 2 , d -Squame Pressure Instrument D500).…”

“…Tapes underwent DNA extraction using a DNeasy blood and tissue kit (Qiagen, Germany) per the manufacturer’s instructions, with a few modifications as previously outlined ( 39 ). Similarly, metabarcoding was performed targeting the universal V3-V4 16S rRNA region of bacteria using the 341F (5′- CCTAYGGGRBGCASCAG -3′) and 806R (5′- GGACTACNNGGGTATCTAAT -3′) primers.…”

“…Similarly, metabarcoding was performed targeting the universal V3-V4 16S rRNA region of bacteria using the 341F (5′- CCTAYGGGRBGCASCAG -3′) and 806R (5′- GGACTACNNGGGTATCTAAT -3′) primers. Sequencing preparation was carried out as fully described by Barnes et al ( 39 ) using a TruSeq DNA PCR-Free Library Preparation Kits (Illumina, CA, USA). Completed libraries were sent for 250-paired end sequencing at the National High-Throughput Sequencing Center of Denmark (University of Copenhagen, Denmark) on an Illumina MiSeq platform (CA, USA).…”

Nutritional and ecological perspectives of the interrelationships between diet and the gut microbiome in multiple sclerosis: Insights from marmosets

“…used Paired-end assembler for Illumina sequences (PANDAseq) for read assembly and Quantitative Insights into Microbial Ecoloby (QIIME) for quality control and analysis, whereas here a pipeline that combines several other publicly available tools was used (Illumina-utils, USEARCH, and QIIME, among others; see Data S1 ). This moderate pipeline-based variation in results is expected ( O'Rourke et al., 2020 ; Bokulich et al., 2018 ; Xue et al., 2018 ; Rajan et al., 2019 ; Barnes et al., 2020 ). Despite these discrepancies in the abundances of select taxa, we emphasize that our re-analysis identified the same suite of dominant taxa in the marmoset microbiome that were originally described by Kap et al., with Actinobacteria , Firmicutes and Bacteroidetes present at high (>10%) relative abundances.…”

PURC v2.0: A Program for Improved Sequence Inference for Polyploid Phylogenetics and Other Manifestations of the Multiple-Copy Problem

“…Since the release of PURC, several studies have demonstrated shortcomings of OTU clustering, which is the method PURC used to infer biological sequences. In particular, OTU clustering tends to overestimate the number of sequences, is sometime unreproducible (repeat runs yield different OTUs), and it is difficult to determine appropriate similarity thresholds (Callahan et al, 2017;Barnes et al, 2020;Joos et al, 2020;Nelson et al, 2020), with the overestimation problem reported for PURC specifically (Morales-Briones and Tank, 2019;Blischak et al, 2018). An alternative approach to identify and separate PCR and sequencing errors from biological sequences is to apply an error model, where read abundance, composition, and quality scores are used to infer whether each unique read is likely to have been derived from another observed sequence (Callahan et al, 2016).…”