©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 20 (1): gmr18757 Resolving a human identification case for the Rio de Janeiro Police with massively parallel sequencing of mtDNA using a proposed pipeline C. Bottino 1,2 , R. Silva 3 and R.S. Moura-Neto 4 1 Instituto de Pesquisa e Perícias em Genética Forense, Brasil 2 Instituto Nacional de Metrologia, Qualidade e Tecnologia, Brasil 3 Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brasil 4 Instituto de Biologia, Universidade Federal do Rio de Janeiro, Brasil Corresponding author: R.S. Moura-Neto E-mail: rodrigomouraneto@ufrj.br Genet. Mol. Res. 20 (1): gmr18757 Received October 25, 2020 Accepted March 10, 2021 Published March 31, 2021 DOI http://dx.doi.org/10.4238/gmr18757 ABSTRACT. The forensic community is concerned about the quality control of mitotypes reported, as probability estimates may be biased to the disadvantage of a suspect when there is incomplete data. This is particularly important when using new technology such as Massively Parallel Sequencing (MPS), which performs simultaneous analysis of millions of DNA fragments, generating vast amounts of data from small amounts of sample, providing more extensive genetic information. The use of MPS for complex forensic evidence analysis could save time and resources, and provide reliable data for extremely degraded DNA samples. However, data analysis methods can be challenging to implement in a forensic labs routine. Here, we propose a reliable and straightforward pipeline for mtDNA data analysis in forensic samples using MPS, and we used this pipeline to solve a human identification case by Rio de Janeiro Police who had recovered a severely putrefied body that could not be identified by conventional autosomal STR analysis. Mitochondrial DNA (mtDNA) from samples were sequenced on Ion Torrent PGM. Raw data from the sequencing was analyzed in two main pipeline protocols: quality control and forensic analysis. The first includes the analyses of raw