title: Analysis of body fluid mixtures by mtDNA sequencing: An inter-laboratory study of the GEP-ISFG working group.
abstract: The mitochondrial DNA (mtDNA) working group of the GEP-ISFG (Spanish and Portuguese Group of the International Society for Forensic Genetics) carried out an inter-laboratory exercise consisting of the analysis of mtDNA sequencing patterns in mixed stains (saliva/semen and blood/semen). Mixtures were prepared with saliva or blood from a female donor and three different semen dilutions (pure, 1:10 and 1:20) in order to simulate forensic casework. All labs extracted the DNA by preferential lysis and amplified and sequenced the first mtDNA hypervariable region (HVS-I). Autosomal and Y-STR markers were also analysed in order to compare nuclear and mitochondrial results from the same DNA extracts. A mixed stain prepared using semen from a vasectomized individual was also analysed. The results were reasonably consistent among labs for the first fractions but not for the second ones, for which some laboratories reported contamination problems. In the first fractions, both the female and male haplotypes were generally detected in those samples prepared with undiluted semen. In contrast, most of the mixtures prepared with diluted semen only yielded the female haplotype, suggesting that the mtDNA copy number per cell is smaller in semen than in saliva or blood. Although the detection level of the male component decreased in accordance with the degree of semen dilution, it was found that the loss of signal was not consistently uniform throughout each electropherogram. Moreover, differences between mixtures prepared from different donors and different body fluids were also observed. We conclude that the particular characteristics of each mixed stain can deeply influence the interpretation of the mtDNA evidence in forensic mixtures (leading in some cases to false exclusions). In this sense, the implementation of preliminary tests with the aim of identifying the fluids involved in the mixture is an essential tool. In addition, in order to prevent incorrect conclusions in the interpretation of electropherograms we strongly recommend: (i) the use of additional sequencing primers to confirm the sequencing results and (ii) interpreting the results to the light of the phylogenetic perspective.
authors.affiliation: Policía Científica, Madrid, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Montesino
authors.forename: A
authors.initials: A
authors.lastname: Salas
authors.forename: M
authors.initials: M
authors.lastname: Crespillo
authors.forename: C
authors.initials: C
authors.lastname: Albarrán
authors.forename: A
authors.initials: A
authors.lastname: Alonso
authors.forename: V
authors.initials: V
authors.lastname: Alvarez-Iglesias
authors.forename: J A
authors.initials: JA
authors.lastname: Cano
authors.forename: M
authors.initials: M
authors.lastname: Carvalho
authors.forename: D
authors.initials: D
authors.lastname: Corach
authors.forename: C
authors.initials: C
authors.lastname: Cruz
authors.forename: A
authors.initials: A
authors.lastname: Di Lonardo
authors.forename: R
authors.initials: R
authors.lastname: Espinheira
authors.forename: M J
authors.initials: MJ
authors.lastname: Farfán
authors.forename: S
authors.initials: S
authors.lastname: Filippini
authors.forename: J
authors.initials: J
authors.lastname: García-Hirschfeld
authors.forename: A
authors.initials: A
authors.lastname: Hernández
authors.forename: G
authors.initials: G
authors.lastname: Lima
authors.forename: C M
authors.initials: CM
authors.lastname: López-Cubría
authors.forename: M
authors.initials: M
authors.lastname: López-Soto
authors.forename: S
authors.initials: S
authors.lastname: Pagano
authors.forename: M
authors.initials: M
authors.lastname: Paredes
authors.forename: M F
authors.initials: MF
authors.lastname: Pinheiro
authors.forename: A M
authors.initials: AM
authors.lastname: Rodríguez-Monge
authors.forename: A
authors.initials: A
authors.lastname: Sala
authors.forename: S
authors.initials: S
authors.lastname: Sóñora
authors.forename: D R
authors.initials: DR
authors.lastname: Sumita
authors.forename: M C
authors.initials: MC
authors.lastname: Vide
authors.forename: M R
authors.initials: MR
authors.lastname: Whittle
authors.forename: A
authors.initials: A
authors.lastname: Zurita
authors.forename: L
authors.initials: L
authors.lastname: Prieto
mesh_terms: D001769:Blood; D002452:Cell Count; D041322:Chromosomes, Human, Y; D019411:Clinical Laboratory Techniques; D016172:DNA Fingerprinting; D004272:DNA, Mitochondrial / Q000235:genetics*; D005260:Female; D006239:Haplotypes; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction; D011786:Quality Control; D012463:Saliva; D012661:Semen; D017422:Sequence Analysis, DNA; D013094:Spermatozoa / Q000166:cytology; D020080:Tandem Repeat Sequences; D014659:Vasectomy
chemical_list: D004272:DNA, Mitochondrial