SAKit: an all-in-one analysis pipeline for identifying novel proteins resulting from variant events at both large and small scales

Background: Genetic mutations that cause the inactivation or aberrant activation of essential proteins may trigger alterations or even dysfunctions in cellular signaling pathways, culminating in the development of precancerous lesions and cancer. Mutations and such dysfunctions can result in the generation of "novel proteins" that are not part of the conventional human proteome. Identification of these proteins carries a profound potential for unraveling promising drug targets and designing innovative therapeutic models. Despite the emergence of diverse tools for detecting DNA or RNA variants, facilitated by the widespread adoption of nucleotide sequencing technology, these methods primarily target point mutations and exhibit suboptimal performance in detecting large-scale and combinatorial mutations. Additionally, the outcomes of these tools are confined to the genome and transcriptome levels, and do not provide the corresponding protein information resulting from genetic alterations. Results: We present the development of Sequencing Analysis Kit (SAKit), a bioinformatics pipeline for hybrid sequencing analysis integrating long-read and short-read RNA sequencing data. Long reads are utilized for detecting large-scale variations such as gene fusions, exon skipping, intron retention, and aberrant expression in non-coding regions, owing to their excellent coverage capabilities. Short reads serve to validate these findings at breakpoints and splice junctions. Conversely, short reads are employed for identifying small-scale variations, including single nucleotide variants, deletions, and insertions, due to their superior sequencing depth, with long reads providing additional validation. SAKit is designed to perform analyses using inter-species configuration files comprising genome references and annotation data, making it applicable to both human and mouse studies. Furthermore, SAKit implements a hierarchical filtering approach to eliminate low-confidence variants and employs open reading frame (ORF) analysis to translate identified variants into protein sequences. Conclusion: SAKit is a robust and versatile bioinformatics tool designed for the comprehensive identification of both large-scale and small-scale variants from RNA-seq data, facilitating the discovery of novel proteins. This pipeline integrates analysis of long-read and short-read sequencing data, offering a powerful solution for researchers in genomics and transcriptomics. SAKit is freely accessible and open-source, available through GitHub (https://github.com/therarna/SAKit) and as a Docker image https://hub.docker.com/repository/docker/therarna). Implemented primarily within a Snakemake framework using Python, SAKit ensures reproducibility, scalability, and ease of use for the scientific community.