T cell receptor repertoires can be profiled using next generation sequencing (NGS) to measure and monitor adaptive dynamical changes in response to disease and other perturbations. Genomic DNA-based bulk sequencing is cost-effective but necessitates multiplex target amplification using multiple primer pairs with highly variable amplification efficiencies. Here, we utilize an equimolar primer mixture and propose a single statistical normalization step that efficiently corrects for amplification bias post sequencing. Using samples analyzed by both our open protocol and a commercial solution, we show high concordance between bulk clonality metrics. This approach is an inexpensive and open-source alternative to commercial solutions.

BackgroundSulfate-reducing bacteria (SRB) drive the ocean sulfur and carbon cycling. They constitute a diverse phylogenetic and physiological group and are widely distributed in anoxic marine environments. From a physiological viewpoint, SRB’s can be categorized as complete or incomplete oxidizers, meaning that they either oxidize their carbon substrate completely to CO2 or to a stoichiometric mix of CO2 and acetate. Members of Desulfofabaceae family are incomplete oxidizers, and within that family, Desulfofaba is the only genus with three isolates that are classified into three species. Previous physiological experiments revealed their capability of respiring oxygen.ResultsHere, we sequenced the genomes of three isolates in Desulfofaba genus and reported on a genomic comparison of the three species to reveal their metabolic potentials. Based on their genomic contents, they all could oxidize propionate to acetate and CO2. We confirmed their phylogenetic position as incomplete oxidizers based on dissimilatory sulfate reductase (DsrAB) phylogeny. We found the complete pathway for dissimilatory sulfate reduction, but also different key genes for nitrogen cycling, including nitrogen fixation, assimilatory nitrate/nitrite reduction, and hydroxylamine reduction to nitrous oxide. Their genomes also contain genes that allow them to cope with oxygen and oxidative stress. They have genes that encode for diverse central metabolisms for utilizing different substrates with the potential for more strains to be isolated in the future, yet their distribution is limited.ConclusionsResults based on marker gene search and curated metagenome assembled genomes search suggest a limited environmental distribution of this genus. Our results reveal a large metabolic versatility within the Desulfofaba genus which establishes their importance in biogeochemical cycling of carbon in their respective habitats, as well as in the support of the entire microbial community through releasing easily degraded organic matters.