Influence of sequencing depth on bacterial classification and abundance in bacterial communities

Microbial diversity is the most abundant form of life. Next Generation Sequencing technologies provide the capacity to study complex bacterial communities, in which the depth and the bioinformatic tools can influence the results. In this work we explored two different protocols for bacterial classification and abundance evaluation, using 10 bacterial genomes in a simulated sample at different sequencing. Protocol A consisted of metagenome assembly with Megahit and Ray Meta and taxonomic classification with Kraken2 and Centrifuge. In protocol B only taxonomicclassification. In both protocols, rarefaction, relative abundance and beta diversity were analyzed. In the protocol A, Megahit had a mean contig length of 1,128 and Ray Meta de 8,893 nucleotides. The number of species correctly classified in all depth assays were 6 out of 10 for protocol A, and 9 out of 10 using protocol B. The rarefaction analysis showed an overestimation of the number of species in almost all assays regardless of the protocol, and the beta diversity analysis results indicated significant differences in all comparisons. Protocol A was more efficient for diversity analysis, while protocol B estimated a more precise relative abundance. Our results do not allow us to suggest an optimal sequencing depth at specie level.

Ant assemblages (Hymenoptera: Formicidae) from areas under the direct influence of two small hydropower plants in Brazil

Current energy production has been the subject of studies on environmental impacts and the need to adequately understand that the relationship to biodiversity loss is growing. One of the ways of assessing environmental changes is the use of bioindicator species, and ants represent an alternative in this regard. This study aimed to evaluate ant assemblages occurring in different environments in areas under the direct influence of two small hydropower plants (SHPP). Sampling was carried out using pitfall traps in forest and agricultural fragments, as well as pasture areas, along the Andrada River, municipality of Cascavel, state of Paraná, in July 2016 and March 2017. The sampled ant assemblages were evaluated for richness, abundance, and composition. The rarefaction analysis was used to compare the richness sampled in the two areas under direct influence. Abundance was analyzed based on the number of occurrences. The nonmetric multidimensional scaling (NMDS) was applied to test whether the abundance and composition of ant assemblages differ at the same site when sampled in both seasons. In total, 63 species belonging to 23 genera and 6 subfamilies were identified. The subfamily Myrmicinae was the most speciesrich (S = 25), followed by the subfamily Formicinae (S = 21). The most species-rich genus was Camponotus (S = 15) followed by Pheidole (S =11). A total of 41.3% richness was registered concurrently in the two assemblages. The study contributes to the expansion of knowledge of the ant fauna occurring in the state of Paraná and serves as a basis for monitoring impacts caused by the implementation of SHPP and other developments.

Metagenome Profiling of Anterior Uterine Samples to Improve in-vitro Fertilization Success Ratio

Background: The radical increase in the number of couples experiencing infertility, which is the inability to conceive in a natural manner, has increased the interest among the researchers worldwide to identify the root cause for this problem. Relevant studies are needed to ascertain the cause behind unexplained infertility, and certain interventions are needed in the process of assisted reproductive technology to improve the implantation success. The presence of certain pathogenic flora in the cervix and vaginal region are often attributed to the failure of In-vitro fertilization (IVF), which is a method of assisted reproduction. This study explored the cervix and vaginal microbiota of 52 such infertile females who underwent assisted reproduction (IVF), to analyse the microbiota, identify pathogenic flora and evaluate the diversity of the flora.Methods: 52 microbial swab samples of infertile females were sequenced using 16s rRNA approach. 16s rRNA data analysis was performed to get the taxonomic abundance and pathogenicity of the resultant microbiota. The microbial communities were statistically assessed for probable similarities and differences between positive, negative and not applicable outcomes of IVF.Results: A total of 31 genera were identified across the study samples, and a thorough investigation of associated pathogenicity of these taxa was performed. Certain pathogenic genera specific to positive as well as negative implantation were observed. Additionally, the alpha diversity was observed to be higher in the cervical region compared to the vaginal region. Rarefaction analysis was performed. The analysis of beta diversity was performed and the phylogenetic distance was visualized through distance boxplots. Conclusion: The presence of pathogenic bacteria, especially those associated with conditions like Bacterial Vaginosis and Chronic Endometritis, are often linked to infertility conditions and were predominant in the cervical and vaginal samples. Dysbiosis in the cervical and vaginal region is often attributed to decrease in Lactobacillus spp. and increase in other species, leading to difference in functionalities and growth of pathogens. In this study, the presence of pathogenic organisms in both regions was remarkable. Additionally, large bacterial diversity in cervical regions was observed, which might be a possible reason for implantation failure. Further studies on the mechanism of action of these bacteria is needed; the possible sources of contamination, such as the tip of the catheter during embryo transfer, the DNA extraction kits prior to sequencing, etc. need to be identified as this might introduce pathogens during the procedure. By using more sterility in the procedure as well as profiling the metagenome of the infertile female, novel insights can be gained as well as a more directed procedure can be performed to increase the chances of success in implantation.

Can DNA help trace the local trade of pangolins? A genetic assessment of white-bellied pangolins from the Dahomey Gap (West Africa)

We conducted in the Dahomey Gap (DG) a pioneer study on the genetic tracing of the African pangolin trade. We sequenced and genotyped 189 white-bellied pangolins from 18 forests and 12 wildlife markets using one mitochondrial fragment and 20 microsatellites loci. Tree-based assignment procedure showed the 'endemicity' of the pangolin trade, as strictly fed by the lineage endemic to the DG (DGL). DGL populations were characterized by low levels of genetic diversity, an overall absence of equilibrium, inbreeding depression and lack of geographic structure. We identified a 92-98% decline in DGL effective population size 200-500 ya –concomitant with major political transformations along the 'Slave Coast' – leading to contemporaneous estimates inferior to minimum viable population size. Genetic tracing suggested that wildlife markets from the DG sourced through the entire DGL range. Our loci provided the necessary power to distinguish among all the genotyped pangolins, tracing the dispatch of same individuals on the markets and within local communities. We developed an approach combining rarefaction analysis of private allele frequencies and cross-validation with observed data that could trace five traded pangolins to their forest origin, c. 200-300 km away from the markets. Although the genetic toolkit that we designed from traditional markers can prove helpful to trace the pangolin trade, our tracing ability was limited by the lack of population structure within DGL. Given the deleterious combination of genetic, demographic and trade-related factors affecting DGL populations, the conservation status of white-bellied pangolins in the DG should be urgently re-evaluated.

18S rRNA amplicon sequence data (V1–V3) of the Bronx river estuary, New York

Characterising and monitoring biological diversity to foster sustainable ecosystems is highly recommended as urban centres rapidly expand. However, much of New York City’s biodiversity remains undescribed, including in the historically degraded, but recovering Bronx River Estuary. In a pilot study to identify organisms and characterise biodiversity patterns there, 18S rRNA gene amplicons (V1–V3 region), obtained from river sediments and surface waters of Hunts Point Riverside and Soundview Parks, were sequenced. Across 48 environmental samples collected over three seasons in 2015 and 2016, following quality control and contaminant removal, 2,763 Amplicon Sequence Variants (ASVs) were identified from 1,918,463 sequences. Rarefaction analysis showed sufficient sampling depth, and community composition varied over time and by substrate at the study sites over the sampling period. Protists, plants, fungi and animals, including organisms of management concern, such as Eastern oysters (Crassostrea virginica), wildlife pathogens and groups related to Harmful Algal Blooms, were detected. The most common taxa identified in river sediments were annelid worms, nematodes and diatoms. In the water column, the most commonly observed organisms were diatoms, algae of the phylum Cryptophyceae, ciliates and dinoflagellates. The presented dataset demonstrates the reach of 18S rRNA metabarcoding for characterising biodiversity in an urban estuary.

Identification of Lagopus muta japonica plant food resources in the Northern Japan Alps using DNA metabarcoding

DNA metabarcoding was employed to identify plant-derived food resources of the Japanese rock ptarmigan ( Lagopus muta japonica ), registered as a natural living monument in Japan, in the Northern Japanese Alps in Toyama Prefecture, Japan, in July to October, 2015-2018. By combined use of rbcL and ITS2 local databases of 74 alpine plant species found in the study area, a total of 43 plant taxa were identified and could be assigned to 40 species (93.0%), two genera (4.7%), and one family (2.3%). Rarefaction analysis of each sample collection period showed that this study covered more than 90% of the plant food resources found in the study area. Of the 21 plant families identified using the combined rbcL and ITS2 local databases, the most dominant families were Ericaceae (98.1% of 105 fecal samples), followed by Rosaceae (42.9%), Apiaceae (35.2%), and Poaceae (19.0%). In all fecal samples examined, the most frequently encountered plant species were Vaccinium ovalifolium var. ovalifolium (69.5%), followed by Empetrum nigrum var. japonicum (68.6%), Vaccinium sp. (54.3%), Kalmia procumbens (42.9%), and Tilingia ajanensis (34.3%). Rarefaction analysis of each collection period in the study revealed that this study covered more than 90% (from 91.0% in July to 97.5% in September) of the plant food resources found in the study area, and 98.1% of the plant food taxa were covered throughout the entire study period. Thus, DNA metabarcoding using the rbcL and ITS2 local databases of alpine plants in combination and rarefaction analysis are considered to be well suited for estimating the dominant food plants in the diet of Japanese rock ptarmigans. Further, the local database constructed in this study can be used to survey other areas with similar flora.

Comprehensive mouse gut metagenome catalog reveals major difference to the human counterpart

Mouse is the most used model for studying the impact of microbiota on its host, but the repertoire of species from the mouse gut microbiome remains largely unknown. Here, we construct a Comprehensive Mouse Gut Metagenome (CMGM) catalog by assembling all currently available mouse gut metagenomes and combining them with published reference and metagenome-assembled genomes. The 50’011 genomes cluster into 1’699 species, of which 78.1% are uncultured, and we discovered 226 new genera, 7 new families, and 1 new order. Rarefaction analysis indicates comprehensive sampling of the species from the mouse gut. CMGM enables an unprecedented coverage of the mouse gut microbiome exceeding 90%. Comparing CMGM to the human gut microbiota shows an overlap 64% at the genus, but only 16% at the species level, demonstrating that human and mouse gut microbiota are largely distinct.

From the Neolithic to the present day: impacts of human presence on biodiversity in the sandstone Northern Vosges (France).

The Northern Vosges and the Pays de Bitche (north-east France) are regions rich in recent industrial inheritance which history is well-known. On the other hand, the ancient history of these regions is not well known and the relationships between human populations and their environment remain unexplored until now for ancient times. The multidisciplinary palaeoenvironmental study that we carried out on the site of the bog-pond located below the ruins of the medieval castle of Waldeck, has made it possible to reconstruct the history of vegetation since 6 600 cal. BP.. Throughout the Holocene, the succession of forest vegetation (pine and hazelnut forests, reduced oak forest, beech forest, oak-beech forest) was largely dominated by pine. The human presence, tenuous during the Neolithic period, is well marked from the Bronze Age onwards with the introduction of crops and livestock crops in the catchment area. From the Middle Ages, anthropic pressure highly increased with the building, in the 13th century, of Waldeck Castle, which led to a major opening of the area. The Modern period is characterized by a gradual return of the forest, while anthropogenic pressure is decreasing. Over time, the occupation phases have been interspersed with abandonment phases during which human activities regress or disappear. Finally, the rarefaction analysis carried out on pollen data shows that human presence has led to a gradual increase in plant diversity, which peaked in the Middle Ages. As a result, the forest has lost some of its resilience to human disturbance over time.

TaxonTableTools - A comprehensive, platform-independent graphical user interface software to explore and visualise DNA metabarcoding data

DNA metabarcoding is increasingly used in research and application to assess biodiversity. Powerful analysis software exists to process raw data. However, when it comes to the translation of sequence read data into biological information many end users with limited bioinformatic expertise struggle with the downstream analysis and explore data only to a minor extent. Thus, there is a growing need for easy-to-use, graphical user interface (GUI) analysis software to analyse and visualise DNA metabarcoding data. We here present TaxonTableTools (TTT), a new platform independent GUI software that aims to fill this gap by providing simple and reproducible analysis and visualisation workflows. TTT uses a so-called “TaXon table” as input. This format can easily be generated within TTT from two input files: a read table and a taxonomy table that can be obtained by various published metabarcoding pipelines. TTT analysis and visualisation modules include e.g. Venn diagrams to compare taxon overlap among replicates, samples or among different analysis methods. It analyses and visualises basic statistics such as read proportion per taxon as well as more sophisticated visualisation such as interactive Krona charts for taxonomic data exploration. Various ecological analyses such as alpha or beta diversity estimates, and rarefaction analysis ordination plots can be produced directly. Data can be explored also in formats required by traditional taxonomy-based analyses of regulatory bioassessment programs. TTT comes with a manual and tutorial, is free and publicly available through GitHub (https://github.com/TillMacher/TaxonTableTools) and the Python package index (https://pypi.org/project/taxontabletools/).

Animal bones in old graves: a zooarchaeological and contextual study on faunal remains and new dated evidence for the ritual re-use of old cemetery sites in Southern and Western Finland

Animal remains from twelve Iron Age (ca. 500 BC–1200/1300AD) sites from Southern and Western Finland, showing a mixture of finds and features typical of both settlement sites and cemeteries, were investigated using a zooarchaeological, taphonomic and contextual approach. Rarefaction analysis of the species richness and anatomical distribution indicates that the samples included both general domestic waste type and species and element-selective deposits of cattle and horse skulls, mandibles and limb bones. According to radiocarbon dating results, there seems to be a gap between the dates of burials and those of other ritual activities, indicating that the context of such deposits is a disused cemetery. The faunal deposits could represent remembrance rituals or relate to votive offerings intended to ensure healthy or productive livestock, a practice described in later ethnographic sources. These deposits seem to be in use within a large geographical area over a long period, and some aspects of this belief system may even have survived into the Christianisation of society in the historical period.