Full mtDNA genome sequencing of Brazilian admixed populations: A forensic-focused evaluation of a MPS application as an alternative to Sanger sequencing methods.

The use of Massive Parallel Sequencing (MPS) techniques have been proposed by the forensic community as an alternative to Sanger sequencing methods in routine forensic casework analysis regarding mitochondrial DNA (mtDNA). Interesting features of MPS include high throughput, ability to simultaneously genotype a significant number of samples by barcoding techniques, processing automation, reduced time and costs, among others. Advantages include the capability of generating full mtDNA genome sequences versus usual techniques, usually limited to hypervariable or control regions exclusively. In this work, 96 reference single-source samples from three different Brazilian cities were subjected to full mtDNA genome sequencing by MPS techniques using an early-access version of Precision ID mtDNA Whole Genome Panel on an Ion Torrent PGM platform (Thermo Fisher Scientific, Waltham, MA, USA). Complete, high-quality sequences were obtained and sequencing performance was evaluated via four different metrics. As a subset of evaluated samples have been previously submitted for Sanger sequencing of the control region, a comparative analysis of both methods' results was conducted in order to compare technique adequacy within a forensic context. Even though this study is one of the first to report full mtDNA genome sequences for Brazilian admixed populations, the observed haplotypes exhibit a predominance of Native American and African maternal lineages in the studied sample set, reproducing results described in the literature for control regions only. Interpopulation analysis among Brazilian and 26 worldwide populations was also carried out. The results indicate that MPS-generated full mtDNA genome sequences may have great utility in forensic real casework applications, with a pronounced gain of genetic information and discrimination power provided by coding region evaluation and the enhanced capacity of heteroplasmies determination. Database construction and other relevant factors concerning implementation of such techniques in Brazilian forensic laboratories are also discussed.

Isolation and characterization of microsatellites from the tapeworm Echinococcus granulosus.

The Echinococcus granulosus genome was searched for microsatellites using 8 different repeated oligonucleotides as probes (GT15, CT15, AT15, CG15, CAT10, CAA10, CGG10 and CATA10). Southern blot experiments revealed that DNA regions containing GT, CAA, CATA and CT repeats are the most frequent in the E. granulosus genome. AT and CG probes showed no hybridization signal. Two loci containing CA/GT (Egmsca1 and Egmsca2) and 1 locus containing GA/CT (Egmsga1) repeats were cloned and sequenced. The locus Egmsca1 was analysed in 73 isolates from Brazil and Argentina whose strains were previously characterized. Brazilian isolates from cattle strain and Argentinean isolates from camel strain were monomorphic and shared the allele (CA)7. Argentinean isolates of sheep and Tasmanian sheep strains shared 2 alleles [(CA)8 and (CA)10] with Brazilian isolates of sheep strain. The allele (CA)11 was found only in Brazilian isolates of sheep strain at a low frequency. The Brazilian and the Argentinean sheep strain populations were tested for the Hardy-Weinberg equilibrium, and only the former was in agreement with the expectations. No polymorphism was found among individual protoscoleces from a single hydatid cyst, validating the utilization of pooled protoscoleces from 1 cyst, grouped as an isolate, in population studies. This work describes for the first time the isolation and characterization of microsatellites from E. granulosus.

Cloning and characterization of Echinococcus granulosus (Cestode) EgactI and EgactII actin gene promoters and their functional analysis in the NIH3T3 mouse cell line

We report here for the first time the structure and function of a promoter from a cestode. The ability of DNA fragments respectively encompassing the 935-bp and 524-bp regions upstream from the ATG codon from the EgactI and EgactII actin genes of Echinococcus granulosus to promote transcription was studied in the NIH3T3 mouse cell line. The results of transfection assays showed that both regions have strong promoter activity in these cells. The fragments were tested in both orientations and the 524-bp fragment of EgactII presented a bidirectional promoter activity. Deletion analysis of EgactI and EgactII promoters indicated the presence of regulatory regions containing putative silencer elements. These results indicate that both EgactI and EgactII promoters are functional and that the preliminary functional evaluation of E. granulosus and possibly of other cestode promoters can be performed in heterologous cell lines

Cloning and characterization of Echinococcus granulosus (Cestode) EgactI and EgactII actin gene promoters and their functional analysis in the NIH3T3 mouse cell line.

We report here for the first time the structure and function of a promoter from a cestode. The ability of DNA fragments respectively encompassing the 935-bp and 524-bp regions upstream from the ATG codon from the EgactI and EgactII actin genes of Echinococcus granulosus to promote transcription was studied in the NIH3T3 mouse cell line. The results of transfection assays showed that both regions have strong promoter activity in these cells. The fragments were tested in both orientations and the 524-bp fragment of EgactII presented a bidirectional promoter activity. Deletion analysis of EgactI and EgactII promoters indicated the presence of regulatory regions containing putative silencer elements. These results indicate that both EgactI and EgactII promoters are functional and that the preliminary functional evaluation of E. granulosus and possibly of other cestode promoters can be performed in heterologous cell lines.