NGSEP is an integrated framework for the analysis of DNA high throughput sequencing data. The main use of this app is the construction and downstream analysis of large datasets of genomic variation. NGSEP performs accurate detection and genotyping of Single Nucleotide Variants (SNVs), small and large indels, short tandem repeats (STRs), inversions, and Copy Number Variants (CNVs). The tool also provides modules for functional annotation, filtering, format conversion, comparison, clustering, imputation, introgression analysis and different kinds of statistics. Other functionalities include calculations of k-mer distributions from fasta or fastq files, demultiplexing of barcoded sequencing reads, and comparative analysis of reading depth distributions.