Additional novel isoforms discovered or validated via the PacBio SMRT sequence data.

<p>Part (a) displays the gene annotation, including the TPR domains (purple) and all alternative splice sites initially discovered by the Illumina RNA-Seq data. Part (b) displays all possible isoforms based on combinations of the major sub-variants plus an extra intron retention between exons 8 and 9b. Part (c) displays the subset of the 624 isoforms that involve one or more “minor” sub-variants (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163590#pone.0163590.g003" target="_blank">Fig 3</a>) that had one or more “perfect” matches in the PacBio SMRT data. Novel features are displayed in pink, and the widened region in each transcript denotes the largest open reading frame (ORF). The data tables on the right side of parts (b) and (c) display the number of full-length ROI from the SMRT sequencing that were found to match each isoform. The first column indicates perfect base-for-base, full-length matches, and the second column lists the total number of matches, including alignment matches in which the RNA-STAR aligner maps the ROI to the isoform. For a complete listing of all possible combinations of all known splice variations see the online supporting information. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163590#pone.0163590.s003" target="_blank">S2 Dataset</a> for more information on these and all other theoretical isoforms.</p

Summary information on the 18 genes detected at p-adjust < 0.0001 in all four analyses.

<p>Column 3 indicates the number of distinct exonic regions (exons), known splice junctions (SJ) and novel splice junctions belonging to this gene, and columns 4–7 indicate the number of each feature type with statistically significant differential usage (# sig, p-adjust < 0.0001). The rightmost column indicates the least significant gene-wise adjusted p-value across the four analyses. *Genes previously known in the literature to exhibit differential isoform regulation between night and day in the rat pineal gland: Crem [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163590#pone.0163590.ref023" target="_blank">23</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163590#pone.0163590.ref025" target="_blank">25</a>], Pde4b [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163590#pone.0163590.ref026" target="_blank">26</a>], and Atp7b [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163590#pone.0163590.ref028" target="_blank">28</a>].</p

Diagram of the 24 potential isoforms produced by the “major” sub-variants, with PacBio match counts.

<p>Part (a) and (b) display the “standard” known isoform and a diagram of the whole gene, respectively. The wide portion of (a) indicates the coding DNA sequence (CDS) of the “standard” transcript, and the purple boxes in (a) and (b) indicate tetratricopeptide repeat (TPR) domains. Part (c) displays the 24 possible combinations formed by the “major” sub-variants from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163590#pone.0163590.g003" target="_blank">Fig 3</a>. Novel features are displayed in pink, and the widened region in each transcript denotes the largest open reading frame (ORF). The data table on the right side of (c) displays the number of full-length ROI from the SMRT sequencing that were found to match each isoform. The first column indicates the number of perfect, base-for-base, full-length matches, and the second column lists “alignment matches”, in which the RNA-STAR aligner maps the ROI to the isoform. Note that the novel start sites were each covered by two primers that were less than 6 base-pairs apart, producing slightly different amplified molecules. Isoforms 21 and 24 were covered by two different primer pairs used in the PCR amplification. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163590#pone.0163590.s004" target="_blank">S3 Dataset</a> for more information on these theoretical isoforms and their PacBio coverage.</p

Results of the start-site qPCR experiment.

<p>Three primer pairs were used, corresponding to the three transcription start sites detected in the Illumina RNA-Seq data. The novel start sites that begin with exons 3a or 3b both display very strong upregulation at night in the control and sham groups.</p

JunctionSeq gene profile plot for Ttc8 gene, sham night/day experiment.

<p>This plot displays the estimates for the mean normalized read-pair coverage count for each exon and splice junction. The small plotting panel on the far right displays the total mean normalized read-pair count for the gene as a whole. The gene diagram below the main plotting frame displays the exonic regions (boxes, labelled E001-E018), known splice junctions (solid lines, labelled J019-J034) and novel splice junctions (dashed lines, labelled N035-N051). Significant features are drawn in pink, and features that did not pass the automatically-selected minimum count threshold were colored in light grey (and were not plotted). Beneath the gene diagram, the genomic positions are marked with ticks at each kilobase. Note that the scale is not uniform due to the nonlinear expansion of small features to improve readability. Beneath the scale the two Ensembl known isoforms are drawn. The upper isoform is the RefSeq transcript.</p

The number of genes detected in each JunctionSeq analysis at various p-value cutoffs.

<p>Also shown is the overlap between the genes detected across multiple separate analyses. Day and night conditions were compared in the four <i>in vivo</i> groups: Control (Ctrl), Sham, decentralized (DCN), and ganglionectomized (SCGX). The two <i>in vitro</i> analyses compared untreated (Untr) pineal glands with pineal glands treated with norepinephrine (NE) or (Untr) vs DBcAMP (DB).</p

Summary browser tracks generated by QoRTs/JunctionSeq for the region surrounding exons 2 to 5 of Ttc8.

<p>The top two tracks display the Ensembl and RefSeq annotations, respectively. The third track displays the names of the exons as they are referred to in the text and in previous literature, with our own names assigned to previously-unidentified exons. The fourth track, generated by QoRTs, displays the mean normalized read-pair coverage depth at each genomic position for night (blue) and day (red) in the sham group. The fifth and sixth tracks, generated by JunctionSeq, display the mean normalized read counts across each known exonic region and across each splice junction (respectively). The bottom track, also generated by JunctionSeq, displays the exons or splice junctions found to display statistically significant differential usage (at p-adjust < 0.01).</p

All possible sub-variants detected in the Illumina RNA-Seq data.

<p>Considering the three major 5’ ends and two major 3’ ends, there are 21 “sub-variants” across 8 “regions”, which can be combined to form 648 different possible combinations. Based on coverage in the Illumina and PacBio data, we narrowed this down to 9 “major” sub-variants that appear to be expressed in the pineal gland in substantial quantities. There are 24 possible combinations of these 9 major sub-variants.</p

Neurotranscriptomics: The Effects of Neonatal Stimulus Deprivation on the Rat Pineal Transcriptome

<div><p>The term neurotranscriptomics is used here to describe genome-wide analysis of neural control of transcriptomes. In this report, next-generation RNA sequencing was using to analyze the effects of neonatal (5-days-of-age) surgical stimulus deprivation on the adult rat pineal transcriptome. In intact animals, more than 3000 coding genes were found to exhibit differential expression (adjusted-p < 0.001) on a night/day basis in the pineal gland (70% of these increased at night, 376 genes changed more than 4-fold in either direction). Of these, more than two thousand genes were not previously known to be differentially expressed on a night/day basis. The night/day changes in expression were almost completely eliminated by neonatal removal (SCGX) or decentralization (DCN) of the superior cervical ganglia (SCG), which innervate the pineal gland. Other than the loss of rhythmic variation, surgical stimulus deprivation had little impact on the abundance of most genes; of particular interest, expression levels of the melatonin-synthesis-related genes Tph1, Gch1, and Asmt displayed little change (less than 35%) following DCN or SCGX. However, strong and consistent changes were observed in the expression of a small number of genes including the gene encoding Serpina1, a secreted protease inhibitor that might influence extracellular architecture. Many of the genes that exhibited night/day differential expression in intact animals also exhibited similar changes following <i>in vitro</i> treatment with norepinephrine, a superior cervical ganglia transmitter, or with an analog of cyclic AMP, a norepinephrine second messenger in this tissue. These findings are of significance in that they establish that the pineal-defining transcriptome is established prior to the neonatal period. Further, this work expands our knowledge of the biological process under neural control in this tissue and underlines the value of RNA sequencing in revealing how neurotransmission influences cell biology.</p></div

Genes significantly differentially expressed in the pineal gland on a night/day basis in Control and/or Sham groups, <i>in vivo</i> (adjusted-p < 0.001).

<p>*Gene exhibits high relative expression in the pineal gland (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137548#pone.0137548.s010" target="_blank">S2 Table</a>).</p><p>^†Gene has previously been found to be differentially expressed between day and night conditions in microarray experiments.</p><p>Genes that exhibit strong differential expression in both are listed in bold (genes are classified by the least-extreme fold change across the two analyses). Note that some genes are listed twice if the fold changes of the two analyses fall in different intervals. In the SCGX and DCN groups, none of the genes displayed in this table exhibited night/day differential expression at this level (adjusted p < 0.001, FC > 4 in either direction). A complete list of all genes with fold changes, p-values, and normalized expression estimates is available in the SI (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137548#pone.0137548.s002" target="_blank">S1 Dataset</a>).</p