Mitochondrial DNA control region typing from highly degraded skeletal remains by single-multiplex next-generation sequencing
Vinueza-Espinosa, Diana C. (Universitat Autònoma de Barcelona. Departament de Biologia Animal, de Biologia Vegetal i d'Ecologia)
Cuesta-Aguirre, Daniel R. (Universitat Autònoma de Barcelona. Departament de Biologia Animal, de Biologia Vegetal i d'Ecologia)
Malgosa Morera, Assumpció (Universitat Autònoma de Barcelona. Departament de Biologia Animal, de Biologia Vegetal i d'Ecologia)
Pereira dos Santos, Cristina Maria (Universitat Autònoma de Barcelona. Departament de Biologia Animal, de Biologia Vegetal i d'Ecologia)

Date:	2023
Abstract:	Poor nuclear DNA preservation from highly degraded skeletal remains is the most limiting factor for the genetic identification of individuals. Mitochondrial DNA (mtDNA) typing, and especially of the control region (CR), using next-generation sequencing (NGS), enables retrieval of valuable genetic information in forensic contexts where highly degraded human skeletal remains are the only source of genetic material. Currently, NGS commercial kits can type all mtDNA-CR in fewer steps than the conventional Sanger technique. The PowerSeq CRM Nested System kit (Promega Corporation) employs a nested multiplex-polymerase chain reaction (PCR) strategy to amplify and index all mtDNA-CR in a single reaction. Our study analyzes the success of mtDNA-CR typing of highly degraded human skeletons using the PowerSeq CRM Nested System kit. We used samples from 41 individuals from different time periods to test three protocols (M1, M2, and M3) based on modifications of PCR conditions. To analyze the detected variants, two bioinformatic procedures were compared: an in-house pipeline and the GeneMarker HTS software. The results showed that many samples were not analyzed when the standard protocol (M1) was used. In contrast, the M3 protocol, which includes 35 PCR cycles and longer denaturation and extension steps, successfully recovered the mtDNA-CR from highly degraded skeletal samples. Mixed base profiles and the percentage of damaged reads were both indicators of possible contamination and can provide better results if used together. Furthermore, our freely available in-house pipeline can provide variants concordant with the forensic software.
Grants:	Agencia Estatal de Investigación PGC2018-096666-B-100 Agència de Gestió d'Ajuts Universitaris i de Recerca 2021/FI_B1 00064 Agència de Gestió d'Ajuts Universitaris i de Recerca 2017/SGR-1630 Agència de Gestió d'Ajuts Universitaris i de Recerca 2021/SGR-00186
Note:	Altres ajuts: acords transformatius de la UAB
Rights:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial i la comunicació pública de l'obra, sempre que no sigui amb finalitats comercials, i sempre que es reconegui l'autoria de l'obra original. No es permet la creació d'obres derivades.
Language:	Anglès
Document:	Article ; recerca ; Versió publicada
Subject:	Ngs ; PowerSeq CRM Nested System kit ; Protocol optimization ; Skeletal samples ; Mtdna
Published in:	Electrophoresis, Vol. 44, Issue 17-18 (September 2023) , p. 1423-1434, ISSN 1522-2683

DOI: 10.1002/elps.202200052
PMID: 37379235

12 p, 796.6 KB

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