Transcription of selected long asRNAs (lasRNAs):

<p>(A) Internalin protein; (B) Internalin protein (note the different scales of x-axis); (C) a novel long antisense transcript with more than 2,400–3,800 nt; (D) predicted SAM-dependent methyltransferase; (E) a rRNA methylase homolog; (F) similar to a methylated DNA protein cystein methyltransferase (note the different scales of x-axis). The upper half of each transcription profile represents the plus strand and the lower one the minus strand. Number of displayed reads is limited to 20. Dark purple – detected ncRNA candidates; lightgreen – NCBI annotation; darkgreen – BacProt annotation; black – reads of the extracellular library; dark blue – reads of the intracellular library; violet – locally stable secondary structure (analyzed with RNALfold); blue – conserved region among other <i>L. monocytogenes</i> serotypes (analyzed with POMAGO); cyan blue – potential new ncRNAs predicted by RNAz; pink – annotated ncRNAs. A better resolution of the figure can be found in the supplement.</p

Comparative analysis of ncRNA transcriptome data:

<p>Comparison of our ncRNA candidates with results of previous studies performed by Toledo-Arana <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108639#pone.0108639-ToledoArana1" target="_blank">[13]</a>, Mraheil <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108639#pone.0108639-Mraheil1" target="_blank">[11]</a> and Wurtzel <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108639#pone.0108639-Wurtzel1" target="_blank">[15]</a>. Note that whenever an ncRNA prediction of this study overlaps with multiple previously described candidates, it is a single hit in the diagram. Altogether, including previous literature, Rfam and this work, now 741 putative ncRNAs are described. In this work we defined 611 to be putative ncRNAs, of which 474 ncRNAs are not part of previous literature, 33 of them known ncRNAs from Rfam.</p

Scoring system.

<p>For evaluation of the ncRNA candidates, a scoring system retrieved from known ncRNAs (Rfam, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108639#pone.0108639-ToledoArana1" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108639#pone.0108639-Wurtzel1" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108639#pone.0108639-Hain4" target="_blank">[26]</a>, see supplemental material) was developed. For increasing length, number of reads and GC content, scores are summed up along the column; for example, an ncRNA candidate of length 100 nt receives a score of +1. The higher the score of a candidate, the higher its probability to be an ncRNA.</p><p>Scoring system.</p

Overview of RNA-seq libraries.

<p>Libraries were retrieved by next generation semiconductor sequencing technology. Number of reads before and after clipping and their mean length.</p><p>Overview of RNA-seq libraries.</p

Selected candidates.

<p>Selected asRNAs and their genomic location, syntenic genes (UpOrf, DownOrf), corresponding GC-content and length (in brackets extended lengths for asRNA detection). IC – number of reads mapped to this region from intracellular library; EC – number of reads mapped to this region from extracellular library; P – number of closely related <i>Listeria</i> serotypes, with a homologous region identified in a genome-wide multiple sequence alignment; RNAz – p-Value of <i>de novo</i> ncRNA prediction of RNAz; RNAL – MFE of locally stable secondary structures, calculated by RNALfold; Score – Score assigned in this study. The complete list of all novel ncRNA candidates can be viewed at the supplemental page.</p><p>Selected candidates.</p

Transcription of a selected long asRNA (lasRNA):

<p><i>kdpABCD</i> operon. Number of displayed reads is limited to 20. Dark purple – detected ncRNA candidates; lightgreen – NCBI annotation; darkgreen – BacProt annotation; black – reads of the extracellular library; dark blue – reads of the intracellular library; violet – locally stable secondary structure (analyzed with RNALfold); blue – conserved region among other <i>L. monocytogenes</i> serotypes (analyzed with POMAGO); cyan blue – potential new ncRNAs predicted by RNAz; pink – annotated ncRNAs; teal green – ncRNA candidates of previous studies.</p

Validation of new long antisense (las) RNAs in <i>L. monocytogenes</i> by qRT-PCR analysis.

<p>(A) The presence of las transcripts was determined by strand-specific qRT-PCR analysis. Supporting the results of RNA-seq, the qRT-PCR analysis indicated that the novel lasRNA transcripts las0333, las0936, las0996, las1136 and las2677 were significantly up-regulated in intracellular conditions. ‘*’ −<i>P</i>≤0.05 ‘**’ −<i>P</i>≤0.01. (B) Strand specific qRT-PCR analysis of las respective target genes shows significant downregulation of <i>lmo0333</i> (internalin), and <i>lmo0936</i> (nitroflavin reductase), upregulation of <i>lmo0996</i> (methyltransferase), <i>lmo1136</i> (internalin) and <i>lmo2677</i> (esterase) in intracellular growth condtions. ‘*’ −<i>P</i>≤0.05; ‘**’ −<i>P</i>≤0.01. Primers used for qRT-PCR are available at the online Supplemental Material.</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

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

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