Evaluating geographic variation within molecular operational taxonomic units (OTUs) using network analyses in Scandinavian lakes

Abstract

Operational taxonomic units (OTUs) are usually treated as if they are internally uniform in environmental metabarcoding studies of microbial and macrobial eukaryotes, even when the OTUs are being used to infer biogeographic patterns. The OTUs constructed by the program Swarm have underlying network topologies in which nodes represent amplicons and edges represent 1 nucleotide differences between nodes. Such networks can be exploited to search for biogeographic patterns within each OTU. To do this, here we used an available protistan metabarcoding dataset consisting of the hypervariable V4 region of the 18S rRNA locus amplified from 77 lakes collected across Norway and Sweden. The 82 most abundant and wide-spread OTUs constructed by Swarm were evaluated using shortest path, assortativity, and geographical analyses. We found that while pairs of amplicons from the same lake were usually connected directly to each other within the OTUs, these pairs of amplicons from the same lake did not form assortative clusters within the OTUs, and amplicons were not more connected with other amplicons occurring in neighboring lakes than expected by chance. This new approach to looking at within-OTU is applicable to other metabarcoding datasets and we provide code to perform these analyses.

Swarm v3: towards tera-scale amplicon clustering

“…Swarm v3 can be used on short and long read metabarcoding data (with sequences up to 10 Mbp when using d = 1), or on meta-transcriptomic/genomic data that has been subsampled from the same locus. It offers a comprehensive set of options that gives users full-control and access to intermediate internal data, such as the complete pairwise sequence network (see Forster et al , 2020 , for a usage example). Swarm v3 is open-source, actively maintained, portable and efficient, thus reducing the need for expensive computational resources.…”

Genetic Diversity in Marine Planktonic Ciliates (Alveolata, Ciliophora) Suggests Distinct Geographical Patterns – Data From Chinese and European Coastal Waters

“…A further subdivision of the European OTUs regarding their affiliation to the two different Longhurst ecological provinces and network analyses considering geographical distances were not performed. A case study on a test dataset of Scandinavian lakes detected no significant patterns on a regional scale (Forster et al, 2020). Therefore, we expanded the spatial scale toward two more distant geographical regions in the current study.…”

“…When the computed assortativity values of the original OTU network exceeded the 95% confidence interval of the 1,000 randomized values, amplicons from the same region were significantly more often connected to each other than expected by chance. The workflow and the bioinformatic commands used have previously been published (Forster et al, 2020) and were run in R Studio v. 3.5.1 (R Core Team, 2018), using the igraph package v. 1.2.2 (Csárdi and Nepusz, 2005).…”

“…Swarm OTUs also grow from a centroid 'seed' sequence, but the sequences are linked to each other if their sequence alignment differs by not more than a given number of nucleotides (usually one nucleotide difference, which is likely to arise through biological processes). The internal structure of a Swarm OTU thus represents a network based on the information which sequences are linked to each other, which may be evaluated by means of graph theory (Forster et al, 2020). Evaluating the properties of the internal network structure distinctively increases the resolution of diversity down to single nucleotide differences and, therefore allows unprecedented insights into genetic diversity and geographical distribution patterns.…”

“…Within these environments, assessments of ciliate diversity and distribution patterns were so far confined to whole communities, genera, morphospecies, or OTUs (Foissner et al, 2008;Doherty et al, 2010;Dolan and Pierce, 2013;Massana et al, 2015;Piredda et al, 2017). In the present study, we apply a novel combination of phylogenetic approaches (Hütter et al, 2020) and network analyses (Forster et al, 2019(Forster et al, , 2020 to achieve an unparalleled increase of resolution down to within-OTU level for investigating geographic distribution patterns and their origin in marine planktonic ciliates. Hence, it overcomes the limitations of previous investigations by employing an increased genetic resolution and a unique sampling strategy that links a high spatiotemporal resolution (regional sampling at several sites, at different daytimes, and in different seasons) with a large geographic distance between the regions examined, namely, the Chinese and European coastal waters.…”