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  • Next Generation Sequencing (NGS)
    Comprehensive dataset of shotgun metagenomes from stratified freshwater lakes and ponds
    Moritz Buck, bioRxiv - 2020
    Abstract
    Stratified lakes and ponds featuring steep oxygen gradients are significant net sources of greenhouse gases and hotspots in the carbon cycle. Despite their significant biogeochemical roles, the microbial communities, especially in the oxygen depleted compartments, are poorly known. Here, we present a comprehensive dataset including 267 shotgun metagenomes from 41 stratified lakes and ponds mainly located in the boreal and subarctic regions, but also including one tropical reservoir and one temperate lake. For most lakes and ponds, the data includes a vertical sample set spanning from the oxic surface to the anoxic bottom layer. The majority of the samples were collected during the open water period, but also a total of 29 samples were collected from under the ice. In addition to the metagenomic sequences, the dataset includes environmental variables for the samples, such as oxygen, nutrient and organic carbon concentrations. The dataset is ideal for further exploring the microbial taxonomic and functional diversity in freshwater environments and potential climate change impacts on the functioning of these ecosystems.
    ISSRseq: an extensible, low-cost, and efficient method for reduced representation sequencing
    Brandon T. Sinn, bioRxiv - 2020
    Abstract
    1. The capability to generate densely sampled single nucleotide polymorphism (SNP) data is essential in diverse subdisciplines of biology, including crop breeding, pathology, forensics, forestry, ecology, evolution, and conservation. However, access to the expensive equipment and bioinformatics infrastructure required for genome-scale sequencing is still a limiting factor in the developing world and for institutions with limited resources. 2. Here we present ISSRseq, a PCR-based method for reduced representation of genomic variation using simple sequence repeats as priming sites to sequence inter-simple sequence repeat (ISSR) regions. Briefly, ISSR regions are amplified with single primers, pooled, and used to construct sequencing libraries with a low-cost, efficient commercial kit, and sequenced on the Illumina platform. We also present a flexible bioinformatic pipeline that assembles ISSR loci, calls and hard filters variants, outputs data matrices in common formats, and conducts population analyses using R. 3. Using three angiosperm species as case studies, we demonstrate that ISSRseq is highly repeatable, necessitates only simple wet-lab skills and commonplace instrumentation, is flexible in terms of the number of single primers used, is low-cost, and can generate genomic-scale variant discovery on par with existing RRS methods that require high sample integrity and concentration. 4. ISSRseq represents a straightforward approach to SNP genotyping in any organism, and we predict that this method will be particularly useful for those studying population genomics and phylogeography of non-model organisms. Furthermore, the ease of ISSRseq relative to other RRS methods should prove useful for those conducting research in undergraduate and graduate environments, and more broadly by those lacking access to expensive instrumentation or expertise in bioinformatics.
    Complete Genomes of Symbiotic Cyanobacteria Clarify the Evolution of Vanadium-Nitrogenase
    Jessica M. Nelson, Genome Biology and Evolution - 2019
    Abstract
    Plant endosymbiosis with nitrogen-fixing cyanobacteria has independently evolved in diverse plant lineages, offering a unique window to study the evolution and genetics of plant–microbe interaction. However, very few complete genomes exist for plant cyanobionts, and therefore little is known about their genomic and functional diversity. Here, we present four complete genomes of cyanobacteria isolated from bryophytes. Nanopore long-read sequencing allowed us to obtain circular contigs for all the main chromosomes and most of the plasmids. We found that despite having a low 16S rRNA sequence divergence, the four isolates exhibit considerable genome reorganizations and variation in gene content. Furthermore, three of the four isolates possess genes encoding vanadium (V)-nitrogenase (vnf), which is uncommon among diazotrophs and has not been previously reported in plant cyanobionts. In two cases, the vnf genes were found on plasmids, implying possible plasmid-mediated horizontal gene transfers. Comparative genomic analysis of vnf-containing cyanobacteria further identified a conserved gene cluster. Many genes in this cluster have not been functionally characterized and would be promising candidates for future studies to elucidate V-nitrogenase function and regulation.
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