Efficient and Quality-Optimized Metagenomic Pipeline Designed for Taxonomic Classification in Routine Microbiological Clinical Tests.

Metagenomics analysis is now routinely used for clinical diagnosis in several diseases, and we need confidence in interpreting metagenomics analysis of microbiota. Particularly from the side of clinical microbiology, we consider that it would be a major milestone to further advance microbiota studies with an innovative and significant approach consisting of processing steps and quality assessment for interpreting metagenomics data used for diagnosis. Here, we propose a methodology for taxon identification and abundance assessment of shotgun sequencing data of microbes that are well fitted for clinical setup. Processing steps of quality controls have been developed in order (i) to avoid low-quality reads and sequences, (ii) to optimize abundance thresholds and profiles, (iii) to combine classifiers and reference databases for best classification of species and abundance profiles for both prokaryotic and eukaryotic sequences, and (iv) to introduce external positive control. We find that the best strategy is to use a pipeline composed of a combination of different but complementary classifiers such as Kraken2/Bracken and Kaiju. Such improved quality assessment will have a major impact on the robustness of biological and clinical conclusions drawn from metagenomic studies.

Expression dynamics of the Medicago truncatula transcriptome during the symbiotic interaction with Sinorhizobium meliloti: which role for nitric oxide?

Medicago truncatula is one of the most studied model plants. Nevertheless, the genome of this legume remains incompletely determined. We used RNA-Seq to characterize the transcriptome during the early organogenesis of the nodule and during its functioning. We detected 37,333 expressed transcription units; to our knowledge, 1,670 had never been described before and were functionally annotated. We identified 7,595 new transcribed regions, mostly corresponding to 5' and 3' untranslated region extensions and new exons associated with 5,264 previously annotated genes. We also inferred 23,165 putative transcript isoforms from 6,587 genes and measured the abundance of transcripts for each isoform, which suggests an important role for alternative splicing in the generation of proteome diversity in M. truncatula. Finally, we carried out a differential expression analysis, which provided a comprehensive view of transcriptional reprogramming during nodulation. In particular, depletion of nitric oxide in roots inoculated with Sinorhizobium meliloti greatly increased our understanding of the role of this reactive species in the optimal establishment of the symbiotic interaction, revealing differential patterns of expression for 2,030 genes and pointing to the inhibition of the expression of defense genes.

Nitric oxide is required for an optimal establishment of the Medicago truncatula-Sinorhizobium meliloti symbiosis.

Nitric oxide (NO) is a gaseous molecule that participates in numerous plant signalling pathways. It is involved in plant responses to pathogens and development processes such as seed germination, flowering and stomatal closure. Using a permeable NO-specific fluorescent probe and a bacterial reporter strain expressing the lacZ gene under the control of a NO-responsive promoter, we detected NO production in the first steps, during infection threads growth, of the Medicago truncatula-Sinorhizobium meliloti symbiotic interaction. Nitric oxide was also detected, by confocal microscopy, in nodule primordia. Depletion of NO caused by cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide), an NO scavenger, resulted in a significant delay in nodule appearance. The overexpression of a bacterial hmp gene, encoding a flavohaemoglobin able to scavenge NO, under the control of a nodule-specific promoter (pENOD20) in transgenic roots, led to the same phenotype. The NO scavenging resulting from these approaches provoked the downregulation of plant genes involved in nodule development, such as MtCRE1 and MtCCS52A. Furthermore, an Hmp-overexpressing S. meliloti mutant strain was found to be less competitive than the wild type in the nodulation process. Taken together, these results indicate that NO is required for an optimal establishment of the M. truncatula-S. meliloti symbiotic interaction.