DNA is a powerful tool available for forensic investigations requiring identification of species. However, it is necessary to develop and validate methods able to produce results in degraded and or low quality DNA samples with the high standards obligatory in forensic research. Here, we describe a voluntary collaborative exercise to test the recently developed Species Identification by Insertions/Deletions (SPInDel) method. The SPInDel kit allows the identification of species by the generation of numeric profiles combining the lengths of six mitochondrial ribosomal RNA (rRNA) gene regions amplified in a single reaction followed by capillary electrophoresis. The exercise was organized during 2014 by a Working Commission of the Spanish and Portuguese-Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG), created in 2013. The 24 participating laboratories from 10 countries were asked to identify the species in 11 DNA samples from previous GHEP-ISFG proficiency tests using a SPInDel primer mix and control samples of the 10 target species. A computer software was also provided to the participants to assist the analyses of the results. All samples were correctly identified by 22 of the 24 laboratories, including samples with low amounts of DNA (hair shafts) and mixtures of saliva and blood. Correct species identifications were obtained in 238 of the 241 (98.8%) reported SPInDel profiles. Two laboratories were responsible for the three cases of misclassifications. The SPInDel was efficient in the identification of species in mixtures considering that only a single laboratory failed to detect a mixture in one sample. This result suggests that SPInDel is a valid method for mixture analyses without the need for DNA sequencing, with the advantage of identifying more than one species in a single reaction. The low frequency of wrong (5.0%) and missing (2.1%) alleles did not interfere with the correct species identification, which demonstrated the advantage of using a method based on the analysis of multiple loci. Overall, the SPInDel method was easily implemented by laboratories using different genotyping platforms, the interpretation of results was straightforward and the SPInDel software was used without any problems. The results of this collaborative exercise indicate that the SPInDel method can be applied successfully in forensic casework investigations.

Delegação do Norte Instituto Nacional de Medicina Legal e Ciências Forenses 1 P Porto Portugal

Forensic Science Unit Forensic Genetics Section Basque Country Police Ertzaintza Erandio Bizkaia Spain

Grupo de Medicina Xenómica Instituto de Ciencias Forenses Universidade de Santiago de Compostela Spain

Institute of Criminalistics Prague Prague Czech Republic

Instituto de Investigação e Inovação em Saúde I3S Universidade do Porto Portugal; Instituto de Patologia e Imunologia Molecular da Universidade do Porto Porto Portugal

Instituto de Investigação e Inovação em Saúde I3S Universidade do Porto Portugal; Instituto de Patologia e Imunologia Molecular da Universidade do Porto Porto Portugal; Faculty of Sciences University of Porto Porto Portugal

Instituto de Medicina Legal de Las Palmas Laboratorio Genética Forense Spain

INTCF Instituto Nacional de Toxicología y Ciencias Forenses Delegación de Canarias Santa Cruz de Tenerife Spain

Interdisciplinary Centre of Marine and Environmental Research University of Porto Porto Portugal

Istituto di Sanità Pubblica Sezione di Medicina Legale Università Cattolica del Sacro Cuore Roma Italy

Laboratorio Biología ADN Comisaría General de Policía Científica Instituto Universitario de Investigación en Ciencias Policiales Spain

Laboratorio de Análisis de ADN Facultad de Ciencias Médicas UNCuyo Mendoza Argentina

Laboratório de Diagnóstico por DNA Instituto de Biologia Roberto Alcântara Gomes Universidade do Estado do Rio de Janeiro Rio de Janeiro Brazil

Laboratório de Genética Aplicada Universidade de Aveiro Portugal

Laboratorio de Genética Molecular Cruz Roja Ecuatoriana Cruz Vital Quito Ecuador

Laboratorio de Genética Molecular Instituto Multidisciplinario de Biología Celular CIC CONICET UNLP La Plata Argentina

Laboratorio del Equipo Argentino de Antropología Forense Córdoba Argentina

Laboratorio Genes SAS Medellín Colombia and Instituto de Biología Universidad de Antioquia Medellín Colombia

PRICAI Fundación Favaloro CABA Argentina

Sección de Bioquímica Departamento de Ciencias Forenses Poder Judicial de Costa Rica Costa Rica

Sección de genética forense y criminalistica Instituto de Medicina Legal y Ciencias Forenses de Valencia Valencia Spain

Servicio de Criminalística de la Guardia Civil Departamento de Química y Medio Ambiente Madrid Spain

Serviço de Genética e Biologia Forense Delegação do Sul Instituto Nacional de Medicina Legal e Ciências Forenses 1 P Lisboa Portugal

Serviço de Genética e Biologia Forenses Delegação do Centro Instituto Nacional de Medicina Legal e Ciências Forenses 1 P Coimbra Portugal

Unidad de Diagnóstico Molecular Departamento de Bioquímica y Medicina Molecular Facultad de Medicina Universidad Autónoma de Nuevo León Monterrey México

Unidad de Laboratorio de Navarra de Servicios y Tecnologías Villava Spain

Universidad de Buenos Aires Facultad de Farmacia y Bioquímica Departamento de Microbiología Inmunología y Biotecnología Cátedra de Genética Forense y Servicio de Huellas Digitales Genéticas Junín 956 and CONICET Consejo Nacional de Investigaciones Científicas y Tecnológicas C1033AAJ Buenos Aires Argentina

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