2.1 Patient recruitment procedure and RNA extraction

A total of 13 samples with low TPM genes from patients with VUSs were identified through the Splicing and Disease Research Study at the University of Southampton, UK, ethics approved by the Health Research Authority (IRAS Project ID 49685, REC 11/SC/0269) and by the University of Southampton (ERGO ID23056). Informed consent for splicing studies was provided for all patients from whom samples were obtained. Patient blood samples were collected using PAXgene Blood RNA tubes and intracellular RNA extraction from whole blood was performed with the PAXgene Blood RNA Kit (PreAnalytix). All the patient RNA sample's quality and concentration were checked with bioanalyzer before performing RT-PCR and RNA-seq.

2.2 RNA-seq and data analysis

Whole transcriptome sequencing with ribosomal RNA depletion and stranded library preparation was carried out via Novogene using NovoSeq 600 PE150. A minimum coverage depth of 70M 150 base pair paired-end reads were produced for each sample. Distribution of sequencing quality, error rate distribution, A/T/G/C distribution, the composition of raw data, and data quality summary were mentioned in the supplementary data (Data S1). Data analysis was performed on the IRIDIS 4 high-performance computer cluster, University of Southampton. FASTQ reads were aligned to the reference human genome version 38 (GRCh38) using the STAR alignment tool (2.5.2b version) (Dobin et al., 2013) with reading level filters: reads corresponding to a mapping quality of 255 and maximum mismatch of 6. These filtering parameters were used to match those employed by GTEx. Filtered aligned BAM files were visualized and sashimi plots were generated using the integrative genomics viewer (Ttir et al., 2013).

2.3 RT-PCR and polymerase chain reaction

All the experiments were conducted in the laboratory of Human Development and Health, Southampton General Hospital, University of Southampton. Complementary DNA (cDNA) was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) using random hexamers. Primer pairs (Data S) were designed using the online Primer3web tool (primer3.ut.ee), selecting binding sites in exons adjacent to variants and standard desalted synthetic single-stranded DNA oligo primers were ordered from Integrated DNA Technologies. Polymerase chain reaction (PCR) experiments were performed using the GoTaq G2 Polymerase PCR system (Promega) and GoTaq Hot Start Polymerase (Promega) according to the manufacturer's protocol. PCR cycles were set up at 95°C for 5 min followed by 35 and 40 repetitive cycles of 95°C for 30 s, 60°C or the annealing temperature of individual primer pairs for 30 s and 72°C for 1 min per kilobase pair before ending the cycle with 72°C for 10 min and cooling down at 4°C. RT-PCR products were purified by GeneJET PCR Purification Kit (Thermo Fisher Scientific) and bidirectional Sanger sequencing was carried out by SourceBioscience. Where indicated, amplicons for further analysis were gel-purified by GeneJET Gel Extraction Kit (Thermo Fisher Scientific) and sequenced at SourceBioscience for Sanger sequencing. All the PCR experiments were repeated at least twice for reproducibility and all expected positive amplicons were confirmed by Sanger sequencing.

3.1 RNA-seq sensitivity is reduced for low TPM genes

TPM is a normalized RNA abundance measurement of high-throughput RNA sequencing data. The values are calculated using the number of reads aligned to a gene of interest and they come from their respective genes or transcripts for every one million RNA molecules in the RNA-seq sample (Wagner et al., 2012). It is a useful indicator of gene expression level and for predicting the detectability of splicing events using reads spanning exon–exon junctions. Exon-spanning reads are crucial for splicing analysis using RNA-seq and these are abundant in genes where the median TPM value is more than 1 (Figure 1). Aberrant splicing events can also be readily visualized in genes where the TPM value is 1 or more in RNA-seq of blood RNA. However, exonic and exon-spanning reads of genes with median TPM below 1 show lower or no read coverage (Figure 1). Therefore, aberrant splicing events can be missed due to insufficient read coverage where TPM values are lower than 1 and cannot be easily analyzed using the RNA-seq.

3.2 Short amplicon RT-PCR is more sensitive than standard RT-PCR in detecting genes with low TPM in the blood

The primers we designed for conventional RT-PCR for the detection of aberrant splicing are located so as to span at least seven exons, where the exon harboring the variant is at the center of the amplicon. In this way, aberrant splicing events such as one or two exon skipping affecting upstream or downstream exons with respect to the variant of interest can be detected by gel electrophoresis. This design of PCR for aberrant splicing detection is sensitive to genes that have high TPM values in the blood (Figure 2a). However, it fails to produce any amplicons for genes that have lower TPM values (less than 1) (Figure 2b–g). On the other hand, a short amplicon RT-PCR design, in which primers span only three exons, is sensitive enough to amplify the targeted cDNA (Figure 2b–g) when performed at 35 cycles using GoTaq G2 DNA polymerase. As only three exons are spanned in short amplicon PCR, only single-exon-skipping aberrant splicing events can be detected and two exon-skipping events may be missed.

Both short and long amplicons were further analyzed using different PCR conditions such as amplification cycles and polymerases. Two different amplification cycles, 35 and 40 cycles, and two different Taq DNA polymerases, GoTaq G2 and GoTaq Hot Start polymerases, were tested for the three genes with low TPM values. Only short amplicons were detected in all different PCR conditions whereas nonspecific amplicons or no amplicons were detected in long amplicon PCR reactions, confirming that short amplicon PCR has higher sensitivity than a long one (Figure S1).

3.3 Short amplicon RT-PCR detects normal and aberrant transcripts of genes with low TPM values

Four different categories of low TPM genes were analyzed using short amplicon RT-PCRs. These categories were based on the GTEx database median TPM values: 1 to 0.1 (COL1A1, COL5A2, DSP, SYN1, TCF4, TTN), 0.1 to 0.01 (NTRK2, TERT), 0.01 to 0.001 (ABCA4), and 0 (PRPH, WT1). Amplicons were successfully detected in genes with median TPM ranges from 1 to 0.001 using short amplicon RT-PCR (Figure 3). Aberrant splicing events were detected in SYN1 c.838-2 A>G (median TMP= 0.8549) (Figure 3a), TCF4 c.550-3C>G (median TPM = 0.4009), NTRK2 c.287+3G>C (median TPM = 0.03444) (Figure 3d), TERT c.3295+5G>T (median TPM = 0.04452), TERT c.5137+3A>G (median TPM = 0.04452) (Figure 3e), TTN c.49346-1G>A (median TPM = 0.2851), and TTN c.63793G>A (median TPM = 0.2851). In comparison, sashimi plots of RNA-seq read coverage data across splice junctions for low TPM gene regions show poor or no exonic or exon-spanning reads covering these areas. The results of all VUSs tested are summarized in Table 1. Amino acid changes caused by aberrant splicing are also shown in Table 1.