Fig. 1

Overview of the FLGSA assay and research strategy. a Representation of the SPINK1 full-length gene expression vector and the experimental steps involved in the FLGSA assay for each study variant. The coding sequences of the four-exon SPINK1 gene are depicted to scale, while the intronic and untranslated region sequences are not. The reference SPINK1 genomic sequence is NG_008356.2, and the reference SPINK1 mRNA sequence is MANE (Matched Annotation from the NCBI and EMBL-EBI [45]) select ENST00000296695 or NM_001379610.1. NM_001379610.1 represents the SPINK1 transcript isoform expressed in the exocrine pancreas [29, 30]. The starting and ending positions of the coding sequences in each exon, as well as those of the SPINK1 genomic sequence cloned into the pcDNA3.1/V5-His-TOPO vector, are indicated in accordance with NM_001379610. b Illustration demonstrating how the FLGSA assay was integrated with SpliceAI to prospectively evaluate the splicing effects of all potential coding variants within the SPINK1 gene. Abbreviations: FLGSA, full-length gene splicing assay; RT-PCR, reverse transcription-PCR; SNVs, single-nucleotide variants