Additional file 1 of Long read sequencing reveals novel isoforms and insights into splicing regulation during cell state changes
Creators: name: Wright, David J.
name: Hall, Nicola A. L.
name: Irish, Naomi
name: Man, Angela L.
name: Glynn, Will
name: Mould, Arne
name: Angeles, Alejandro De Los
name: Angiolini, Emily
name: Swarbreck, David
name: Gharbi, Karim
name: Tunbridge, Elizabeth M.
name: Haerty, Wilfried
Publisher: name: figshare
Year: 2022
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Figure S1. Schematic representation of the custom annotation pipeline, utilising TALON software (Wyman et al. 2020) with custom bash, python and perl auxiliary and processing scripts (collated in clean_TALON_output.pl, see script repository). Fig. S2. Schematic representation of CACNA2D2 (ENSG00000007402) transcripts, showing the novel transcript TALONT000703030. Figure modified from IsoformSwitchAnalyzeR output (Vitting-Seerup and Sandelin 2019). Fig. S3. Short read (Illumina paired-end) coverage plot of novel first exon (31 bp) of CACNA2D2 (ENSG00000007402) transcript TALONT000703030 from all 10 sequencing runs (see Table S1). Fig. S4. Coverage plot of novel first exon (31 bp) of CACNA2D2 (ENSG00000007402) transcript TALONT000703030 from N = 27 human cortex RNA-seq GTEx accessions from N = 21 individuals: SRR1310008, SRR1311400, SRR1311575, SRR1315866, SRR1316815, SRR1317344, SRR1320963, SRR1323043, SRR1326179, SRR1331579, SRR1333930, SRR1337564, SRR1339651, SRR1343481, SRR1353176, SRR1354446, SRR1364676, SRR1368772, SRR1382732, SRR1383059, SRR1387809, SRR1418837, SRR1418992, SRR1433971, SRR1435293, SRR1444580, SRR1468514. Fig. S5. Comparison of an example annotated coding transcript (ENST00000479441) of CACNA2D2 (ENSG00000007402) with the novel transcript TALONT000703030, demonstrating key differences and initial 3D structure rendered using Phyre2 (Kelley et al. 2015). Fig. S6. Schematic representation of primer placement for RT-PCR validation of the novel CACNA2D2 exon and transcript (TALON000703030) relative to two representative examples of previously known transcripts. Note, the same reverse primer is used for each forward. See Table S5 for primer details. Fig. S7. Gel electrophoresis image of the CACNA2D2 RT-PCR validation. Each primer set is labelled corresponding to Table S5 and Fig S6, along with the negative and positive controls. Fig. S8. Custom UCSC Genome Browser visualization of the full coverage of short read (pink) and long read RNA-Seq (blue) across the CACNA2D2 genome model for a single sample (differentiated cells; sample D2). Read peaks supporting the novel exon shown on far right of tracks. Table S1. Summary statistics of the Oxford Nanopore Technologies (ONT) after quality checking and Illumina paired-end short read sequencing (SRS) of 10 replicate samples of human neuronal cell line SH-SY5Y. D = differentiated cell and U = undifferentiated cell samples. Table S2. Comparison of limit of quantification (LOQ) of Oxford Nanopore Technologies (ONT) sequencing, Illumina short read sequencing (SRS) and Illumina reads downsampled to average nucleotide coverage of ONT reads. LOQ calculated by Anaquin (Wong et al. 2017). Table S3. Differential gene and transcript expression at a stringent filter of logFC ⋛ 1.5, FDR q-value