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Ultra deep sequencing of Listeria monocytogenes sRNA transcriptome revealed new antisense RNAs

Listeria monocytogenes, a gram-positive pathogen, and causative agent of listeriosis, has become a widely used model organism for intracellular infections. Recent studies have identified small non-coding RNAs (sRNAs) as important factors for regulating gene expression and pathogenicity of L. monocytogenes. Increased speed and reduced costs of high throughput sequencing (HTS) techniques have made RNA sequencing (RNA-Seq) the state-of-the-art method to study bacterial transcriptomes. We created a large transcriptome dataset of L. monocytogenes containing a total of 21 million reads, using the SOLiD sequencing technology. The dataset contained cDNA sequences generated from L. monocytogenes RNA collected under intracellular and extracellular condition and additionally was size fractioned into three different size ranges from 150 nt. We report here, the identification of nine new sRNAs candidates of L. monocytogenes and a reevaluation of known sRNAs of L. monocytogenes EGD-e. Automatic comparison to known sRNAs revealed a high recovery rate of 55%, which was increased to 90% by manual revision of the data. Moreover, thorough classification of known sRNAs shed further light on their possible biological functions. Interestingly among the newly identified sRNA candidates are antisense RNAs (asRNAs) associated to the housekeeping genes purA, fumC and pgi and potentially their regulation, emphasizing the significance of sRNAs for metabolic adaptation in L. monocytogenes

Next Generation Gene Synthesis by targeted retrieval of bead-immobilized, sequence verified DNA clones from a high throughput pyrosequencing device

The setup of synthetic biological systems involving millions of bases is still limited by the required high quality of synthetic DNA. Important drivers to further open up the field are the accuracy and scale of chemical DNA synthesis and the downstream processing of longer DNA assembled from short fragments. We developed a new, highly parallel and miniaturized method for the preparation of high quality DNA termed “Megacloning” by using Next Generation Sequencing (NGS) technology in a preparative way. We demonstrate our method by processing both conventional and microarray-derived DNA oligonucleotides in combination with a bead-based high throughput pyrosequencing platform, gaining a 500-fold error reduction for microarray oligonucleotides in a first embodiment. We also show the assembly of synthetic genes as part of the Megacloning process. In principle, up to millions of DNA fragments can be sequenced, characterized and sorted in a single Megacloner run, enabling many new applications

Intervening in return to work after long term sickness absence : confronting the stakeholders views

Background Acknowledging individual and societal consequences of long term sickness absence, a political awareness is raising in Belgium in favour of a more active reintegration policy. Since various stakeholders are implied and several legislations may apply, it was deemed necessary to analyse the interplay between regulations and the role of the respective interveners to optimise return to work practices. The study focused on workers who are still under contract. Methods To this purpose, 23 representatives of various categories of stakeholders were interviewed: social insurance physicians, occupational physicians, insurers, social administration services, employers, unions, employment advisers, etc… Interviewees were asked to describe their role, to identify difficulties in the execution of their mission and sources of conflict between legislations, and to propose improvements. The interviews were audiotaped and fully transcribed. Results Several barriers to worker reintegration were pointed out during the interviews. On individual level, the worker social situation and a lack of information influence the chance of a successful return to work. At the enterprise level, the enterprise size, quality of peer support and lack of financial incentives were often mentioned. Most remarks concerned legislation. In Belgian labour law, the employment contract may be ended if the employee is permanently unable to perform his current job, even though modified work would remain possible. For many stakeholders, a sustainable return to work is hindered by the complexity of legislation which was developed for different domains (disability benefits, unemployment, work accident, occupational disease) with little attention to transitions from one domain to the other, especially between benefit insurance and unemployment legislation. It was also stressed that the possibilities offered by the occupational health legislation are underused or inappropriately used. Conclusions Legal and financial security must be insured for the worker who is ready to enrol in a reintegration trajectory. Public authorities should promote better knowledge and implementation of reintegration policies and current legislation should be improved with an emphasis on a better collaboration between stakeholders.Retour au travail après une absence de longue duré

Pileup of reads representing the TSS of the <i>dnaA</i> gene of <i>L. monocytogenes</i>.

<p>Reads are mapped onto the <i>L. monocytogenes</i> genome and depicted as horizontal lines in the top half of the figure. Forward reads are mapped above, reverse reads below the base line. Blue reads are from the sample containing RNA fragments <40 nt, green reads from the sample containing RNA between 40 nt and 150 nt, red reads from the fraction containing RNA >150 nt. The lower half of the figure shows the corresponding annotation at this genome location, with the beginning of the <i>dnaA</i> gene at position 318. Artemis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083979#pone.0083979-Rutherford1" target="_blank">[39]</a> was used to illustrate the mapped reads and annotation of the genome.</p

Pileup of reads representing four newly identified asRNAs of <i>L. monocytogenes</i>.

<p>Putative sRNAs are marked with red boxes. Each colored line represents a mapped read either on the forward strand (above the line) or the reverse strand (below the line). Blue reads are from the sample containing RNA fragments <40 nt, green reads from the sample containing RNA between 40 and150 nt. Red reads from the sample of RNAs >150 nt. The lower half of each figure shows the corresponding annotation at this genome location. (A) anti0055 (<i>purA</i>). Shown is the extracellular condition. (B) anti2225 (<i>fumC</i>). Shown is the extracellular condition. (C) anti2330 (<i>lmo2331</i>) in phage locus of <i>L. monocytogenes</i>. Shown is the extracellular condition. (D) anti2367 (<i>pgi</i>). Shown is the intracellular and extracellular condition respectively. Expression of the <i>pgi</i> gene and the boxed antisense RNA is mutual exclusive between the two conditions.</p

List of the nine newly identified sRNAs in <i>L. monocytogenes</i>, which are classified as asRNAs with the corresponding antisense gene given.

<p>List of the nine newly identified sRNAs in <i>L. monocytogenes</i>, which are classified as asRNAs with the corresponding antisense gene given.</p

sRNAs identified by different studies [2]–[4] and this study and their overlap. sRNAs for this study were identified via automatic identification with our newly developed pipeline.

<p>144 (55%) known sRNAs were recovered with the automated method. Of the 711 sRNAs identified in total, 569 were yet undescribed. The majority of these, however, were later removed due to their likely origin as transcription start site and 5′ UTR of known genes. Most of sRNAs, which were not recalled by the automated method, were found by manual reevaluation, increasing the total recall rate to 90%.</p

Validation of new asRNA transcripts from <i>L.</i> monocytogenes and their effect on gene regulation after transition to the intracellular growth conditions.

<p>A) The antisense RNA transcript anti0055 (<i>purA</i>) is validated by northern blot analysis and strand-specific qRT-PCR. The graph shows intracellular up-regulation of anti0055. B) Northern blot images of anti0055 and control 5S rRNA EC: Extracellular, IC: Intracellular. C) The presence of antisense transcripts anti2106 (<i>lmo2106</i>), anti2225 (<i>fumC</i>), and anti2330 (<i>lmo2330</i>) was determined by strand-specific qRT-PCR. anti2330 is down-regulated, anti2106 and anti2225 are up-regulated significantly. D) Strand-specific qRT-PCR analysis confirmed the existence and up-regulation of antisense RNA transcript anti2367. <i>pgi</i> (<i>lmo236</i>7) was down-regulated, which indicates the possible role of anti2367 in <i>pgi</i> gene regulation. ‘*’ P≤0.05; ‘**’ P≤0.01; ‘***’ P≤0.001.</p

Schematic representation of the main computational pipeline used in this study and its input and output.

<p>The pipeline is optimized to work with sequence data from fractionated RNA samples containing RNA fragments of different lengths. Data gathered under various conditions can also be used for differential expression analysis. For this study we used data from the SOLiD High Throughput Sequencing (HTS) platform, but the pipeline will also process data from all major HTS platforms. The individual steps within the pipeline are colored either gray or orange representing steps for which existing software was used and newly implemented features respectively. The result of the pipeline will be lists of pre-classified sRNA candidates.</p