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A 2b-RAD parentage analysis pipeline for complex and mixed DNA samples

NCJ Number
Forensic Science International: Genetics Volume: 55 Issue: 102590 Dated: 2021
Date Published

This article describes the authors’ research into next-generation DNA sequencing technology, in which they discuss the following topics: the limitations of parentage analysis based on microsatellite markers; 2b-RAD cost effectiveness, with low DNA inputs, and robust to DNA degradation; their development of a probabilistic genotyping-by-sequencing genetic testing pipeline; their detection of biases in closely related or inbred individuals; and they note that their approach resolves mixed DNA samples.


Next-generation sequencing technology has revolutionized genotyping in many fields of study, yet parentage analysis often still relies on microsatellite markers that are costly to generate and are currently available only for a limited number of species. 2b-RAD sequencing (2b-RAD) is a DNA sequencing technique developed for ecological population genomics that utilizes type IIB restriction enzymes to generate consistent, uniform fragments across samples. This technology is inexpensive, effective with low DNA inputs, and robust to DNA degradation. In their research, the authors developed a probabilistic genotyping-by-sequencing genetic testing pipeline for parentage analysis by using 2b-RAD for inferring familial relationships from mixed DNA samples and populations. Their approach to partial paternity assignment utilizes a novel weighted outlier paternity index (WOPI) adapted for next-generation sequencing data and an identity-by-state (IBS) 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 parentage when closely related or inbred individuals are in the alleged parent population (e.g., in laboratory strains); and 2) it resolves mixed DNA samples. The authors successfully demonstrate this novel approach by correctly inferring paternity for samples pooled from multiple offspring (i.e., entire clutches) in a highly inbred population of an East African cichlid fish. The unique advantages of 2b-RAD in combination with their bioinformatics pipeline enable straightforward and cost-effective parentage analysis in any species regardless of genomic resources available. Publisher Abstract Provided

Date Published: January 1, 2021