Identification of body fluids by mRNA analysis with minion nanopore sequencing

WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
Mary Jessamine Michaels (Creator)
Institution
Western Carolina University (WCU )
Web Site: http://library.wcu.edu/
Advisor
Kelly Grisedale

Abstract: The identification of body fluids present on evidence items in a criminal investigation can be vital to understanding the nature of a crime, particularly in cases of sexual assault. Although crime labs can confirm the presence of body fluids like semen and blood on a piece of evidence using traditional serological techniques, they cannot confirm the presence of saliva or vaginal fluid or differentiate peripheral blood from menstrual blood. Due to the unique patterns of gene expression in different cell types, different body fluids contain distinct messenger RNA (mRNA) molecules, which can be analyzed to generate mRNA profiles for confirmatory identification of body fluids. High throughput sequencing methods present the opportunity to generate large amounts of data from low level forensic samples that is not achievable with more traditional techniques. In this study, the MinION sequencer by Oxford Nanopore Technologies (ONT), which is a small and affordable high throughput sequencer that generates data rapidly and in real time, was assessed to determine its ability to generate high quality data from forensic type samples. Semen, saliva, blood, vaginal fluid, and menstrual blood from eight different donors were collected on sterile swabs. Additionally, a tenfold dilution series was performed on semen, blood, and saliva samples from three donors each and decreasing volumes of semen were pipetted onto vaginal swabs from three sets of donors. DNA and RNA were co-extracted from half swabs. DNA fractions were taken forward for short tandem repeat (STR) analysis. For RNA fractions, cDNA was generated, and a multiplex PCR targeting two genes per body fluid was performed. Amplicons were sequenced on the MinION with the 1D Ligation Sequencing Kit by ONT. Full DNA profiles were obtained for all semen, blood, and menstrual blood samples, and most of the saliva and vaginal fluid samples. Profiles were obtained for diluted semen, saliva, and blood samples, and mixed profiles were obtained for the semen/vaginal fluid mixture samples. The multiplex PCR was highly specific for each body fluid, with little to no cross reactivity. The MinION was able to obtain at least 1,000X coverage of target genes and little to no off target reads, even for some diluted samples. Both vaginal fluid and semen genes were detected in mixture samples. Optimization of the MinION sequencing workflow to maximize read counts and minimize costs should be explored. Despite the high input requirements stated by ONT for sequencing, the MinION appears to be able to generate high quality data with lower DNA/RNA input.

Additional Information

Publication
Thesis
Language: English
Date: 2018
Keywords
body fluid identification, forensic, MinION, mRNA, nanopore sequencing

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