RESUMO
The purpose of this paper is to formulate recommendations for the disclosure of biological traces in the laboratory and the handling of forensic evidence submitted for identification tests, recommended by the Polish Speaking Working Group of the International Society for Forensic Genetics. The paper organizes the knowledge of the most relevant stages of preliminary analysis of biological traces based on both literature sources and those resulting from years of research practice. Recommendations formulated in the course of multi-stage expert consultations contained in this study should be used in the development of laboratory procedures applied during the execution.
Assuntos
Genética Forense , Humanos , Polônia , Genética Forense/normas , Genética Forense/métodos , Genética Forense/legislação & jurisprudência , Sociedades Científicas/normas , Impressões Digitais de DNA/normas , Revelação/normas , Revelação/legislação & jurisprudênciaRESUMO
Massively parallel sequencing (MPS) technology has become the gold standard in mitochondrial DNA research due to its high sensitivity in detecting mtDNA heteroplasmy, a prognostic marker in various medical applications. Various MPS technologies and platforms used for mtDNA analysis exist. Obtaining reliable and sensitive results requires deep and uniform coverage of the entire mtDNA sequence, which is heavily influenced by the choice of library preparation method and sequencing platform. Here, we present a comparison of the sequencing coverage and the ability to heteroplasmy detection using two library preparation protocols (Nextera XT DNA Library Preparation Kit and Nextera DNA Flex Library Preparation Kit) and two different (MiSeq FGx and ISeq 100) Illumina MPS platforms. Our study indicates that the Nextera DNA Flex Library protocol provides a more balanced coverage along the mitogenome and a reliable heteroplasmy detection with both MiSeq and iSeq Illumina MPS systems.
Assuntos
DNA Mitocondrial , Biblioteca Gênica , Heteroplasmia , Sequenciamento de Nucleotídeos em Larga Escala , DNA Mitocondrial/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Heteroplasmia/genética , Análise de Sequência de DNA/métodos , Genoma Mitocondrial/genéticaRESUMO
Introduction: Massively parallel sequencing of mitogenomes usually requires prior amplification. The PCR step may influence the quality of the data obtained, especially when low-level heteroplasmy detection is applied. Aim: The aim of this study was to compare the reliability of two different DNA polymerases in detecting homoplasmic and heteroplasmic substitutions in human mitogenomes. Material and methods: Mitogenomes of five samples were amplified with Long PCR Enzyme Mix from Fermentas or TaKaRa LA Taq DNA Polymerase from TaKaRa. Then, NexteraTM XT DNA libraries were sequenced on MiSeq FGx platform (Illumina). mtDNA substitutions were called for alternative variants above the 1% level. Results: All homoplasmic substitutions detected in amplicons generated with polymerases studied here and sequenced on MiSeq FGx system were consistently identified as homoplasmies with alternative sequencing methods. TaKaRa LA Taq DNA Polymerase was found to be less accurate in low-level heteroplasmy detection than Long PCR Enzyme Mix enzyme as more false negative and false positive results were observed for minority variants called above the 1% level. Nevertheless, both PCR systems studied can be successfully used to detect authentic mtDNA substitutions, for which minority variants exceed the 3.61% level assuming at least 10,000x coverage and sequencing Nextera XT DNA libraries on MiSeq FGx machine. Conclusions: The accuracy and sensitivity of point heteroplasmy detection with the MiSeq FGx instrument varies on polymerase used for mtDNA amplification. Therefore, it is recommended to validate the laboratory protocols used for mtDNA substitution detection prior to their implementation for the forensic or medical genetics purposes.