Seq2pathway is a novel tool for functional gene-set (or termed as pathway) analysis of next-generation sequencing data, consisting of "seq2gene" and "gene2path" components. The seq2gene links sequence-level measurements of genomic regions (including SNPs or point mutation coordinates) to gene-level scores, and the gene2pathway summarizes gene scores to pathway-scores for each sample. The seq2gene has the feasibility to assign both coding and non-exon regions to a broader range of neighboring genes than only the nearest one, thus facilitating the study of functional non-coding regions. The gene2pathway takes into account the quantity of significance for gene members within a pathway compared those outside a pathway. The output of seq2pathway is a general structure of quantitative pathway-level scores, thus allowing one to functional interpret such datasets as RNA-seq, ChIP-seq, GWAS, and derived from other next generational sequencing experiments.