A comparison between SOLiD 5500XLand Ion Torrent PGM-derived miRNA expression profiles in two breast cell lines.

Next-generation sequencing (NGS) platforms allow the analysis of hundreds of millions of molecules in a single sequencing run, revolutionizing many research areas. NGS-based microRNA studies enable expression quantification in unprecedented scale without the limitations of closed-platforms. Yet, whereas a massive amount of data produced by these platforms is available, comparisons of quantification/discovery capabilities between platforms are still lacking. Here we compare two NGS-platforms: SOLiD and PGM, by evaluating their microRNA identification/quantification capabilities using two breast-derived cell-lines. A high expression correlation (R2 > 0.9) was achieved, encompassing 97% of the miRNAs, and the few discrepancies in miRNA counts were attributable to molecules that have very low expression. Quantification divergences indicative of artefactual representation were seen for 14 miRNAs (higher in SOLiD-reads) and another 10 miRNAs more abundant in PGM-data. An inspection of these revealed an increased and statistically significant count of uracyls and uracyl-stretches for PGM-enriched miRNAs, compared to SOLiD and to the miRBase. In parallel, adenines and adenine-stretches were enriched for SOLiDderived miRNA reads. We conclude that, whereas both platforms are overall consistent and can be used interchangeably for microRNA expression studies, particular sequence features appear to be indicative of specific platform bias, and their presence in microRNAs should be considered for database-analyses.

Identification of DNA mutations in gastric washes from gastric adenocarcinoma patients: Possible implications for liquid biopsies and patient follow-up.

Whereas cancer patients have benefited from liquid biopsies, the scenario for gastric adenocarcinoma (GAC) is still dismal. We used next-generation deep sequencing of TP53-a highly mutated and informative gene in GAC-to assess mutations in tumor biopsies, plasma (PL) and stomach fluids (gastric wash-GW). We evaluated their potential to reveal tumor-derived mutations, useful for monitoring mutational dynamics at diagnosis, progression and treatment. Exon-capture libraries were constructed from 46 patients including tumor biopsies, GW and PL pre and post-treatment (196 samples), with high vertical coverage >8,000×. At diagnosis, we detected TP53 mutations in 15/46 biopsies (32.6%), 7/46 GW- (15.2%) and 6/46 PL-samples (13%). Biopsies and GW were concordant in 38/46 cases (82.6%) for the presence/absence of mutations and, furthermore, four GW-exclusive mutations were identified, suggesting tumor heterogeneity. Considering the combined analysis of GW and PL, TP53 mutations found in biopsies were also identified in 9/15 (60%) of cases, the highest detection level reported for GAC. Our study indicates that GW could be useful to track DNA alterations, especially if anchored to a comprehensive gene-panel designed for this malignancy.

Detection of Schistosoma mansoni infection by TaqMan® Real-Time PCR in a hamster model.

An experimental study in hamsters was performed to evaluate the capability for detecting Schistosoma mansoni DNA in serum and fecal samples during the pre and post-egg-laying periods of infection using TaqMan® Real-Time PCR system (qPCR), was compared with the circumoval precipitin test (COPT) and the Kato-Katz technique, especially among individuals with low parasitic burden. Twenty-four hamsters were infected with cercariae. Three hamsters were sacrificed per week under anesthesia, from 7 days post infection (DPI) up to 56 DPI. A serum sample and a pool of feces were collected from each hamster. The presence of S. mansoni eggs in fecal samples was evaluated by Kato-Katz method and in the hamsters gutby histopathology. Detection of S. mansoni DNA was performed using qPCR and S. mansoni antibody using COPT. The first detection of eggs in feces by Kato-Katz method and S. mansoni DNA in feces by qPCR occurred 49 DPI. Nevertheless, S. mansoni DNA was detected in serum samples from 14 up to 56 DPI. COPT was positive at 35 DPI. The results not only confirm the reliability of S. mansoni DNA detection by qPCR, but also demonstrate that serum is a trustworthy source of DNA in the pre patent infection period.

Exome sequencing of multiple-sclerosis patients and their unaffected first-degree relatives.

OBJECTIVES: The understanding of complex multifactorial diseases requires the availability of a variety of data for a large-number of affected individuals. In this data note here we provide whole exome sequencing data from a set of non-familiar multiple-sclerosis (MS) patients as well as their unaffected first-degree relatives. This data might help the identification of genomic alterations, including single nucleotide polymorphisms, de novo variations and structural genomic variations, such as copy-number alterations that may impact this disease. DATA DESCRIPTION: This dataset comprises the full exome of 28 Brazilian subjects grouped in eight distinct families, consisting of four complete trios (mother-patient-father) plus another four complete trios with one added unaffected sibling. In total, we present the full exome data of eight patients diagnosed with recurrent remittent multiple sclerosis. Diagnoses were made by experienced neurologists and all enrolled patients had at least 5 years of follow up and specific MS treatment. Exomes were sequenced from leukocyte-derived DNA, after the capture of exons using biotinylated probes, in the Ion Proton platform. For each exome we generated an average of 66.1 million good quality mapped reads with an average length of ~ 160nt. On average, for 90% of the exome a vertical coverage above 20× was reached.

Estudo de vesículas extracelulares em linhagens celulares e empacientes com câncer de mama positivas para a amplificação deERBB2/HER2 / Study of extracellular vesicles in cell lines and breast cancer patients positive for ERBB2/HER2 amplification

As Vesículas Extracelulares (EVs) são organelas essenciais para processos fisiológicos e aparentemente contribuem para o desenvolvimento de diversas doenças através da transferência entre células de ácidos nucléicos e proteínas contidos em seu interior. Deste modo, EVs são promissoras como fonte de biomarcadores. Neste trabalho, avaliamos o conteúdo de EVs secretadas in vitro e in vivo, com o intuito de identificar moléculas relacionadas com a amplificação do oncogene ERBB2/HER2 e com estádios de progressão tumoral de câncer de mama HER2+. Utilizando um modelo de duas linhagens celulares - HB4a derivada de mama normal e C5.2, um clone de HB4a que superexpressa o oncogene ERBB2/HER2 - isolamos duas populações de EVs provenientes do meio de cultura celular condicionado: 20K, enriquecida para microvesículas e 100K, enriquecida para exossomos. Demonstramos que a superexpressão de um único oncogene (ERBB2/HER2) altera o perfil proteômico das EVs secretadas pela células C5.2. Proteínas capazes de induzir a transformação maligna foram encontradas superexpressas nas duas populações de EVs da linhagem C5.2, e interessantemente observamos uma maior expressão de HER2 nas EVs em relação a esta mesma linhagem celular. Uma análise por sequenciamento de nova geração do transcriptoma completo (RNAs pequenos e longos) das EVs e células secretoras revelou centenas de transcritos diferencialmente representados entre as duas linhagens, que podem estar direta ou indiretamente sendo afetados pela superexpressão de HER2, e possibilitou uma investigação das moléculas preferencialmente incorporadas nas EVs...

Extracellular vesicles (EVs) are essential organelles involved in physiological processes and apparently contribute to the development of several pathological conditions by the transfer between cells of nucleic acid and protein cargo. Therefore, EVs are promising sources of biomarkers. In the study presented here, we evaluated the content of EVs secreted in vitro and in vivo, aiming to identify molecules related to ERBB2/HER2 oncogene amplification and with progressing tumor stages of HER2+ breast cancer. Using a model of two cell lines – HB4a derived from normal breast and C5.2, a clone of HB4a that overexpresses ERBB2/HER2 – we isolated two populations of EVs derived from the conditioned cell culture medium: 20K, enriched for microvesicles and 100K, enriched for exosomes. We demonstrate that the overexpression of a single oncogene (ERBB2/HER2) alters the proteomic landscape of EVs secreted by C5.2 cells. Proteins capable of inducing malignant transformation were found overexpressed in both EVs populations secreted by C5.2 cells, and interestingly, we observed an increased expression of HER2 in the EVs compared to the secreting cells. Next generation sequencing analysis of the entire transcriptome (long and short RNAs) of EVs and secreting cells revealed hundreds of transcripts differentially expressed between the two cell lines, which may be directly or indirectly affected by HER2 overexpression, and enabled the investigation of molecules that are preferentially shuttled in EVs...