Keywords

Forensic DNA Analysis, Spermatozoa, Direct Single Cell Subsampling, Extended Interval Post-Coital Samples, Mixture Deconvolution, Haploid Interpretation

Abstract

Following a sexual assault, the ability to obtain an autosomal short tandem repeat (aSTR) profile of a semen contributor diminishes rapidly and is often not possible for intervals exceeding 48-72 hours. This partially stems from limitations with DNA extraction and typing systems used. Routine analysis typically uses differential extraction (i.e. separating sperm and non-sperm cells). This can make fragile sperm prematurely lyse, causing the sperm DNA to become overwhelmed by the amount of victim epithelial DNA. While Y-chromosome STRs (Y-STRs) target male DNA despite abundant female DNA, Y-STR haplotypes are less discriminatory than aSTR profiles. To address these issues, this work optimized single sperm DNA profiling methods for use with challenging post-coital samples. Single sperm were collected and analyzed directly, avoiding sperm loss while eliminating admixtures. Direct single cell subsampling (DSCS) was paired with epithelial cell depletion and amplification conditions suitable for low template DNA samples, to recover single source aSTR haplotypes from samples collected at post-coital intervals where typically only Y-STR haplotype recovery is possible. This included intervals up to 143-hours post-coital (US recommended collection interval is 120-hours). These aSTR profiles had random match probabilities (i.e. statistical likelihood that evidence profile will match a random, unrelated person in a population) up to 1 in 12.3 billion (compared to Y-STR haplotype frequency for the same donor of 1 in 106,445). This was applied to 2-, 3-, and 4-person semen mixtures. These haploid samples were used to develop clustering methods suitable for reconstituting donor profiles with probabilistic genotyping replicate analysis (i.e. statistical modeling for complex DNA profile interpretation). These clusters yielded highly probative diploid genotypes, suitable for offender database searching. This capability to isolate aSTR iv haplotypes and reconstruct probative diploid genotypes highlights the significant potential of this approach to deconvolute complex sexual assault samples that would otherwise yield nondiscriminatory results.

Completion Date

2025

Semester

Fall

Committee Chair

Hanson, Erin

Degree

Doctor of Philosophy (Ph.D.)

College

College of Sciences

Department

Chemistry

Format

PDF

Identifier

DP0029755

Document Type

Thesis

Campus Location

Orlando (Main) Campus

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