Unraveling the Stratification of an Iron-Oxidizing Microbial Mat by Metatranscriptomics

International audienceA metatranscriptomic approach was used to study community gene expression in a naturally occurring iron-rich microbial mat. Total microbial community RNA was reversely transcribed and sequenced by pyrosequencing. Characterization of expressed gene sequences provided accurate and detailed information of the composition of the transcriptionally active community and revealed phylogenetic and functional stratifications within the mat. Comparison of 16S rRNA reads and delineation of OTUs showed significantly lower values of metatranscriptomic-based richness and diversity in the upper parts of the mat than in the deeper regions. Taxonomic affiliation of rRNA sequences and mRNA genome recruitments indicated that iron-oxidizing bacteria affiliated to the genus Leptothrix, dominated the community in the upper layers of the mat. Surprisingly, type I methanotrophs contributed to the majority of the sequences in the deep layers of the mat. Analysis of mRNA expression patterns showed that genes encoding the three subunits of the particulate methane monooxygenase (pmoCAB) were the most highly expressed in our dataset. These results provide strong hints that iron-oxidation and methane-oxidation occur simultaneously in microbial mats and that both groups of microorganisms are major players in the functioning of this ecosystem

Impact of the severity of negative energy balance on gene expression in the subcutaneous adipose tissue of periparturient primiparous Holstein dairy cows: Identification of potential novel metabolic signals for the reproductive system

The severity of negative energy balance (NEB) in high-producing dairy cows has a high incidence among health diseases. The cow's energy status during early lactation critically affects metabolic and reproductive parameters. The first objective of this study was to investigate by RNA-seq analysis and RT-qPCR the gene expression profile in white adipose tissue and by gene ontology and upstream regulation tools the relationships with energy metabolism and reproduction in two groups of primiparous dairy cows with extreme NEB statuses (NEB < -9 Mcal/day vs. NEB > -9 Mcal/day) around parturition. The second objective was to determine the potential involvement of a new adipokine identified as a candidate for the regulation of ovarian function in our RNA-seq analysis by using bovine primary granulosa culture, thymidine incorporation to determine cell proliferation and ELISA assays to measure progesterone secretion. The RNA-seq analysis revealed that 514 genes were over-expressed and 695 were under-expressed in the adipose tissue of cows with severe NEB (SNEB) and cows with moderate NEB (MNEB) during the -4 and 16 wkpp period. In addition, 491 genes were over-expressed and 705 genes were under-expressed in the adipose tissue of SNEB cows compared to MNEB cows. Among these differently expressed genes (DEGs), 298 were related to metabolic functions and 264 to reproductive traits. A set of 19 DEGs were validated by RT-qPCR, including CCL21 (C-C motif chemokine ligand 21). Moreover, CCL21, a gene known to be secreted by adipose tissue, was chosen for further analysis in plasma and ovaries. The use of next-generation sequencing technologies allowed us to characterise the transcriptome of white adipose tissue from primiparous cows with different levels of NEB during lactation. This study highlighted the alteration of the expression of genes related to lipid metabolism, including CCL21, which is released in the bloodstream and associated with the in vitro regulation of ovarian functions

Assemblathon 2: evaluating de novo methods of genome assembly in three vertebrate species

Background: The process of generating raw genome sequence data continues to become cheaper, faster, and more accurate. However, assembly of such data into high-quality, finished genome sequences remains challenging. Many genome assembly tools are available, but they differ greatly in terms of their performance (speed, scalability, hardware requirements, acceptance of newer read technologies) and in their final output (composition of assembled sequence). More importantly, it remains largely unclear how to best assess the quality of assembled genome sequences. The Assemblathon competitions are intended to assess current state-of-the-art methods in genome assembly. Results: In Assemblathon 2, we provided a variety of sequence data to be assembled for three vertebrate species (a bird, a fish, and snake). This resulted in a total of 43 submitted assemblies from 21 participating teams. We evaluated these assemblies using a combination of optical map data, Fosmid sequences, and several statistical methods. From over 100 different metrics, we chose ten key measures by which to assess the overall quality of the assemblies. Conclusions: Many current genome assemblers produced useful assemblies, containing a significant representation of their genes and overall genome structure. However, the high degree of variability between the entries suggests that there is still much room for improvement in the field of genome assembly and that approaches which work well in assembling the genome of one species may not necessarily work well for another

CIRCUS: a package for Circos display of structural genome variations from paired-end and mate-pair sequencing data

International audienceBackgroundDetection of large genomic rearrangements, such as large indels, duplications or translocations is now commonly achieved by next generation sequencing (NGS) approaches. Recently, several tools have been developed to analyze NGS data but the resulting files are difficult to interpret without an additional visualization step. Circos (Genome Res, 19:1639–1645, 2009), a Perl script, is a powerful visualization software that requires setting up numerous configuration files with a large number of parameters to handle. R packages like RCircos (BMC Bioinformatics, 14:244, 2013) or ggbio (Genome Biol, 13:R77, 2012) provide functions to display genomic data as circular Circos-like plots. However, these tools are very general and lack the functions needed to filter, format and adjust specific input genomic data.ResultsWe implemented an R package called CIRCUS to analyze genomic structural variations. It generates both data and configuration files necessary for Circos, to produce graphs. Only few R pre-requisites are necessary. Options are available to deal with heterogeneous data, various chromosome numbers and multi-scale analysis.ConclusionCIRCUS allows fast and versatile analysis of genomic structural variants with Circos plots for users with limited coding skills

source_numerical rawdata_revised

Source numerical raw data</p

Construction of a Transposon Mutant Library in the Pathogen Agrobacterium tumefaciens C58 and Identification of Genes Involved in Gall Niche Exploitation and Colonization

International audienceAgrobacterium tumefaciens is a plant pathogen that causes crown gall disease on a wide range of host species by transferring and integrating a part of its own DNA (T-DNA) into the plant genome. The genes responsible of the above-mentioned processes are well characterized. However, a large number of the mechanisms involved in exploitation and colonization of the galls (also named plant tumors) remain unknown. Due to recent development of “transposon-sequencing” (Tn-Seq) techniques, a high-throughput screening and identification of the different genes involved in such mechanisms is now possible. In this chapter, we describe the detailed methodology used to construct a transposon library in A. tumefaciens and to conduct a Tn-Seq approach to discover genes involved in plant tumor exploitation and colonization

The Third Revolution in Sequencing Technology

International audienceForty years ago the advent of Sanger sequencing was revolutionary as it allowed complete genome sequences to be deciphered for the first time. A second revolution came when next-generation sequencing (NGS) technologies appeared, which made genome sequencing much cheaper and faster. However, NGS methods have several drawbacks and pitfalls, most notably their short reads. Recently, third-generation/long-read methods appeared, which can produce genome assemblies of unprecedented quality. Moreover, these technologies can directly detect epigenetic modifications on native DNA and allow whole-transcript sequencing without the need for assembly. This marks the third revolution in sequencing technology. Here we review and compare the various long-read methods. We discuss their applications and their respective strengths and weaknesses and provide future perspectives

First Complete Genome Sequence of a Salmonella enterica subsp. enterica Serovar Derby Strain Associated with Pork in France

In France, Salmonella enterica subsp. enterica serovar Derby is one of the most often isolated serovars in pigs. Here, we describe the draft genome sequence of a strain isolated from a pig. This strain had the most frequent pulsed-field gel electrophoresis (PFGE) and antimicrobial patterns (S, SSU, T) usually observed in pig production in France. Those patterns have been also highlighted in human isolates