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

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

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

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

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

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