Alternative splicing (AS)
We used the JunctionSeq (Hartley and Mullikin, 2016)pipeline in order to detect genome-wide patterns of alternative
spliced genes. AS is defined as the relative regulation of isoforms
belonging to a multi-isoform gene with respect to a given biological
condition (Hartley and Mullikin, 2015). The pipeline is based on
differential usage calculated from both exon and junction feature
coverages. The pipeline relies on the originally implemented method inDEXSeq (Anders et al., 2012), which tested differential usage of
annotated exons, but extended to splice junction usage and both
annotated and non-annotated splicing events. A new flattened GTF
annotation file where overlapping features are not allowed was first
generated using QoRTs (Hartley and Mullikin, 2015). All
overlapping genes were merged as composed by a flat set of
non-overlapping exons and splice junctions with unique identifiers.QoRTs was also used to generate a read count matrix for AS
analysis, including three types of read counts per gene as estimated by
exons, junction and gene level counts. The generated count matrix was
then used by JunctionSeq R package (Hartley and Mullikin, 2016)to estimate differential exon and junction usage with respect to
gene-wide expression. No read was counted more than once in the model
since exon and junction dispersions are fitted independently. As for DE
analyses, AS genes were detected if at least one exon or splice junction
was differentially used as a result of parental or offspring acclimation
at 36 °C vs 25 °C for TGP and WGP, respectively, using three biological
replicates. Only features with p-values of < 0.05 after FDR
correction were considered significant.