Development of a 2bRAD-seq Paternity Testing Pipeline for Complex and Mixed DNA Samples

As next-generation sequencing technologies have become ever more powerful and cost-effective, they have rapidly replaced short tandem repeat (STR) based techniques in many fields of study. The transition of genetic testing in forensic science will need to handle realistically challenging scenarios such as differentiating highly related individuals and dealing with DNA mixtures. 2b-RAD sequencing is a popular DNA sequencing technique in 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 I propose to develop an automated bioinformatics pipeline for advancing paternity testing by using 2b-RAD sequencing in an ideally challenging genetically-homogenous model laboratory system. This research aims to develop statistical methods 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. The techniques developed include a novel approach to partial paternity assignment, relative combined paternity index adapted for next-generation sequencing data, and an identity-by-state matrix-based clustering method for pedigree reconstruction. The powerful pairing of these two parentage assignment methods allows detection of any biases that might arise from closely related individuals in the alleged parent population and will handle pooled samples of multiple offspring. Successful implementation will aim to advance two major obstacles faced by other genetic testing methods, namely distinguishing parentage of closely related individuals and dealing with mixed-DNA samples. The integration of a bioinformatics pipeline and the unique advantages of 2b-RAD sequencing will allow for relatively easy expansion both into alternative DNA sequencing approaches and any species regardless of restrictive genomic tools, including agricultural systems. Combining and expanding on existing statistical paternity testing and ecological genomics methods provides insight and a foundation for future research in genetic testing. Note: This project contains a research and/or development component, as defined in applicable law," and complies with Part 200 Uniform Requirements - 2 CFR 200.210(a)(14). ca/ncf