Figure 3
Figure 3. RNA-Seq in SS identifies novel Sézary cell-associated transcripts. (A) Graphical representation of the bioinformatic filters used to identify SeCATs. Previously unannotated transcripts were filtered by the pipeline shown to identify 13 candidates that met the criteria noted. (B) Coding potential of known and novel transcripts. Coding potential was measured using the CPC as well as PhyloCFS for 13 novel transcripts. The scores for selected canonical annotated protein-coding and noncoding transcripts are also shown for reference. (C) Expression and conservation of SeCATs. Elemental phastCons conservation across a given transcript (purple; interval between 0 and 0.2 is shaded in yellow, scores > 0.2 are considered conserved), basewise phyloP conservation (green; orange lines = S.D. > average genomic phyloP score), and expression log2 fold change (red = increase, blue = decrease) for 13 SeCATs. Each line on the outside of the circle depicts a SeCAT transcript and is drawn to scale; for reference, the transcript length of SeCAT-10 is 1.1 kb. (D) Relative expression of 13 SeCATs across RNA-Seq human tissue datasets normalized to average RPKM in polyclonal CD4+ T-cells. Gray boxes indicate no detectable expression. Name color indicates directionality of expression change in SC (red = increased, blue = decreased). (E) Genomic locus of SeCAT-7, a novel transcript that is down-regulated in the SC of all 3 patients. Histograms for 1 representative patient have been normalized to account for differences in the number or reads per library (top). The transcript structure predicted by Trinity as well as an unstudied cDNA clone with predicted coding potential and closest flanking genes are shown. Expression levels of SeCAT-7 in normal lymphocytes and SC in each of the 3 patients sequenced. Expression is shown as RPKM and calculated by Scripture (bottom left). Expression of SeCAT-7 by qPCR in CTCL cell lines (bottom right). The average value of CD4+ T-cells from 3 normal donors was used as control. (F) Expression levels of SeCAT-1, 5, 6, and 11 in normal lymphocytes and SC in each of the 3 patients sequenced is shown as RPKM and calculated by Scripture. Expression of SeCAT-1, 5, 6, and 11 is also demonstrated by qPCR in CTCL cell lines. The average value of CD4+ T-cells from 3 normal donors was used as control.

RNA-Seq in SS identifies novel Sézary cell-associated transcripts. (A) Graphical representation of the bioinformatic filters used to identify SeCATs. Previously unannotated transcripts were filtered by the pipeline shown to identify 13 candidates that met the criteria noted. (B) Coding potential of known and novel transcripts. Coding potential was measured using the CPC as well as PhyloCFS for 13 novel transcripts. The scores for selected canonical annotated protein-coding and noncoding transcripts are also shown for reference. (C) Expression and conservation of SeCATs. Elemental phastCons conservation across a given transcript (purple; interval between 0 and 0.2 is shaded in yellow, scores > 0.2 are considered conserved), basewise phyloP conservation (green; orange lines = S.D. > average genomic phyloP score), and expression log2 fold change (red = increase, blue = decrease) for 13 SeCATs. Each line on the outside of the circle depicts a SeCAT transcript and is drawn to scale; for reference, the transcript length of SeCAT-10 is 1.1 kb. (D) Relative expression of 13 SeCATs across RNA-Seq human tissue datasets normalized to average RPKM in polyclonal CD4+ T-cells. Gray boxes indicate no detectable expression. Name color indicates directionality of expression change in SC (red = increased, blue = decreased). (E) Genomic locus of SeCAT-7, a novel transcript that is down-regulated in the SC of all 3 patients. Histograms for 1 representative patient have been normalized to account for differences in the number or reads per library (top). The transcript structure predicted by Trinity as well as an unstudied cDNA clone with predicted coding potential and closest flanking genes are shown. Expression levels of SeCAT-7 in normal lymphocytes and SC in each of the 3 patients sequenced. Expression is shown as RPKM and calculated by Scripture (bottom left). Expression of SeCAT-7 by qPCR in CTCL cell lines (bottom right). The average value of CD4+ T-cells from 3 normal donors was used as control. (F) Expression levels of SeCAT-1, 5, 6, and 11 in normal lymphocytes and SC in each of the 3 patients sequenced is shown as RPKM and calculated by Scripture. Expression of SeCAT-1, 5, 6, and 11 is also demonstrated by qPCR in CTCL cell lines. The average value of CD4+ T-cells from 3 normal donors was used as control.

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