Importance of quality control in sample preparation workflow of forensic degraded samples for massively parallel sequencing (MPS)

Abstract

Molecular genetic analyzes occupy a significant part of laboratory tests in forensic practice. Pre-prepared commercial kits used for DNA extraction are an excellent choice for samples provided by living people, but in forensic practice, most of the samples that undergo molecular processing are often degraded and may pose a challenge in the laboratory. A special challenge is to provide quality genetic material from post-mortem samples that would be suitable for further analysis with massively parallel sequencing. The aim of this paper is to evaluate the applicability of two silica-based DNA extraction methods for post-mortem blood samples with varying storage time and degrees of degradation, and also, to establish proper quality control in sample preparation workflow for Massively Parallel Sequencing using Ion Torrent GeneStudio™ S5 platform. The study was performed on 48 blood samples with different storage time. Silica-based DNA extraction protocols was applied and quantification was made using Qubit 3.0 fluorimeter, followed by Real-Time PCR quantification. The results indicate high variability between the obtained DNA quantities from post-mortem blood samples with different storage time. Correlation was determined between storage time and sample quantity and quality. The Qiagen Mini Kit & Micro Kit are applicable for DNA extraction from blood samples with various storage periods and degradation levels, which can be used for further NGS analyses after careful quality control and optimization of library preparation conditions.