From dual nucleic acid co-extraction to co-sequencing: A highly integrated next-generation forensic DNA and RNA sequencing experimental workflow

DNA and RNA markers are significant in forensic practices, such as individual and body fluid identification. However, forensic DNA and RNA markers were separately analyzed in most forensic experiments, which resulted in large amounts of sample consumption, complex procedures, and weak inter-evidence correlation. While several integrated methods based on capillary electrophoresis and next-generation sequencing technologies were reported, integrated procedures were mostly on nucleic acid co-extraction, co-electrophoresis, or co-sequencing, and the number and type of markers co-tested were limited. Four studies were conducted in this work to co-analyze DNA and RNA with a highly integrated next-generation sequencing-based workflow. First, six commercial kits for DNA and RNA co-extraction were compared, and two kits more suitable for total nucleic acid extraction were selected. Second, the feasibility of an experimental methodology for reverse transcription with total nucleic acid was investigated, and the results showed that reverse transcription reactions have no significant impact on DNA fragments used for forensic short tandem repeat and single nucleotide polymorphism genotyping. Third, a method for DNA and RNA library co-preparation was developed. Through the three studies, a highly integrated experimental workflow for targeted next-generation DNA and RNA sequencing was developed, including DNA and RNA co-extraction, reverse transcription with total nucleic acid, library co-preparation with genomic DNA and complementary DNA, co-sequencing, and data analysis. Fourth, to evaluate the workflow, we developed a multiplex panel consisting of 55 DNA markers (10 autosomal short tandem repeats and 45 autosomal single nucleotide polymorphisms) and eight messenger RNA markers (two peripheral blood-specific, four saliva-specific, and two housekeeping markers), and carried out validation experiments. High accuracy of DNA genotypes and RNA expression results were observed. Taken together, the novel integrated workflow could be used to sequence forensic DNA and RNA markers, which provides a promising method to comprehensively reveal DNA and RNA bioinformation with limited crime scene bio-materials.