As next-generation sequencing technologies have become ever more powerful and affordable, they have begun to replace short tandem repeats (STRs) for genotyping in many fields of study. 2bRAD sequencing (2bRAD-seq) is a DNA sequencing technique developed for ecological population genomics that utilizes type-2b restriction enzymes to generate consistent, uniform fragments across samples. This technology is relatively inexpensive, effective with low DNA inputs, robust, and reliable. Here, we developed a bioinformatics pipeline for advancing paternity testing by using 2bRAD-Seq for interpreting related individuals in DNA mixtures in conjunction with probabilistic genotyping-by-sequencing genetic testing while dealing with additional constraints of high-degree of inbreeding, limited genomic resources, and need to create sibling DNA mixtures. We present a novel approach to partial paternity assignment, which utilizes a relative combined paternity index adapted for next-generation sequencing data and an identity-by-state matrix-based clustering method for pedigree reconstruction. The combination of these two parentage assignment methods overcomes two major obstacles faced by other genetic testing methods: 1) It allows detection of any biases that might arise from closely related or inbred individuals in the alleged parent population (e.g., in laboratory strains); and 2) it can successfully resolve pooled (i.e., mixed) DNA samples of multiple offspring (e.g., when sampling entire clutches). The unique advantages of 2bRAD-Seq allow for straightforward expansion to other species regardless of genomic resources available.