Search Results (9)

Genotypic testing is often recommended to improve the management of patients infected with human immunodeficiency virus (HIV). To help combat this major pandemic, next-generation sequencing (NGS) techniques are widely used to analyse resistance to antiretroviral drugs. In this study, we used a Vela Sentosa kit (Vela Diagnostics, Kendall, Singapore), which is usually used for the Ion Torrent personal genome machine (PGM) platform, to sequence HIV using the Illumina Miseq platform. After RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR), minor modifications were applied to the Vela Sentosa kit to adapt it to the Illumina Miseq platform. Analysis of the results showed the same mutations present in the samples using both sequencing platforms. The total number of reads varied from 185,069 to 752,343 and from 642,162 to 2,074,028 in the Ion Torrent PGM platform and the Illumina Miseq platform, respectively. The average depth was 21,955 and 46,856 for Ion Torrent PGM and Illumina Miseq platforms, respectively. The cost of sequencing a run of eight samples was quite similar between the two platforms (about USD 1790 for Illumina Miseq and about USD 1833 for Ion Torrent PGM platform). We have shown for the first time that it is possible to adapt and use the Vela Sentosa kit for the Illumina Miseq platform to obtain high-quality results with a similar cost. Full article

Fipronil is a broad-spectrum insecticide with remarkable efficacy that is widely used to control insect pests around the world. However, its extensive use has led to increasing soil and water contamination. This fact is of concern and makes it necessary to evaluate the risk of undesirable effects on non-target microorganisms, such as the microbial community in water and/or soil. Studies using the metagenomic approach to assess the effects of fipronil on soil microbial communities are scarce. In this context, the present study was conducted to identify microorganisms that can biodegrade fipronil and that could be of great environmental interest. For this purpose, the targeted metabarcoding approach was performed in soil microcosms under two environmental conditions: fipronil exposure and control (without fipronil). After a 35-day soil microcosm period, the 16S ribosomal RNA (rRNA) gene of all samples was sequenced using the ion torrent personal genome machine (PGM) platform. Our study showed the presence of Proteobacteria, Actinobacteria, and Firmicutes in all of the samples; however, the presence of fipronil in the soil samples resulted in a significant increase in the concentration of bacteria from these phyla. The statistical results indicate that some bacterial genera benefited from soil exposure to fipronil, as in the case of bacteria from the genus Thalassobacillus, while others were affected, as in the case of bacteria from the genus Streptomyces. Overall, the results of this study provide a potential contribution of fipronil-degrading bacteria. Full article

Molecular profiling of a tumor allows the opportunity to design specific therapies which are able to interact only with cancer cells characterized by the accumulation of several genomic aberrations. This study investigates the usefulness of next-generation sequencing (NGS) and mutation-specific analysis methods for the detection of target genes for current therapies in non-small-cell lung cancer (NSCLC), metastatic colorectal cancer (mCRC), and melanoma patients. We focused our attention on EGFR, BRAF, KRAS, and BRAF genes for NSCLC, melanoma, and mCRC samples, respectively. Our study demonstrated that in about 2% of analyzed cases, the two techniques did not show the same or overlapping results. Two patients affected by mCRC resulted in wild-type (WT) for BRAF and two cases with NSCLC were WT for EGFR according to PGM analysis. In contrast, these samples were mutated for the evaluated genes using the therascreen test on Rotor-Gene Q. In conclusion, our experience suggests that it would be appropriate to confirm the WT status of the genes of interest with a more sensitive analysis method to avoid the presence of a small neoplastic clone and drive the clinician to correct patient monitoring. Full article

Studying the means of dispersal of plant pathogens is crucial to better understand the dynamic interactions involved in plant infections. On one hand, entomologists rely mostly on both traditional molecular methods and morphological characteristics, to identify pests. On the other hand, high-throughput sequencing (HTS) is becoming the go-to avenue for scientists studying phytopathogens. These organisms sometimes infect plants, together with insects. Considering the growing number of exotic insect introductions in Canada, forest pest-management efforts would benefit from the development of a high-throughput strategy to investigate the phytopathogenic fungal and oomycete species interacting with wood-boring insects. We recycled formerly discarded preservative fluids from the Canadian Food Inspection Agency annual survey using insect traps and analysed more than one hundred samples originating from across Canada. Using the Ion Torrent Personal Genome Machine (PGM) HTS technology and fusion primers, we performed metabarcoding to screen unwanted fungi and oomycetes species, including Phytophthora spp. Community profiling was conducted on the four different wood-boring, insect-attracting semiochemicals; although the preservative (contained ethanol) also attracted other insects. Phytopathogenic fungi (e.g., Leptographium spp. and Meria laricis in the pine sawyer semiochemical) and oomycetes (mainly Peronospora spp. and Pythium aff. hypogynum in the General Longhorn semiochemical), solely associated with one of the four types of semiochemicals, were detected. This project demonstrated that the insect traps’ semiochemical microbiome represents a new and powerful matrix for screening phytopathogens. Compared to traditional diagnostic techniques, the fluids allowed for a faster and higher throughput assessment of the biodiversity contained within. Additionally, minimal modifications to this approach would allow it to be used in other phytopathology fields. Full article

Fast and affordable benchtop sequencers are becoming more important in improving personalized medical treatment. Still, distinguishing genetic variants between healthy and diseased individuals from sequencing errors remains a challenge. Here we present VARIFI, a pipeline for finding reliable genetic variants (single nucleotide polymorphisms (SNPs) and insertions and deletions (indels)). We optimized parameters in VARIFI by analyzing more than 170 amplicon-sequenced cancer samples produced on the Personal Genome Machine (PGM). In contrast to existing pipelines, VARIFI combines different analysis methods and, based on their concordance, assigns a confidence score to each identified variant. Furthermore, VARIFI applies variant filters for biases associated with the sequencing technologies (e.g., incorrectly identified homopolymer-associated indels with Ion Torrent). VARIFI automatically extracts variant information from publicly available databases and incorporates methods for variant effect prediction. VARIFI requires little computational experience and no in-house compute power since the analyses are conducted on our server. VARIFI is a web-based tool available at varifi.cibiv.univie.ac.at. Full article

Genomic studies performed through liquid biopsies widely elucidated the evolutionary trajectory of RAS mutant clones under the selective pressure of EGFR inhibitors in patients with wild type RAS primary colorectal tumors. Similarly, the disappearance of RAS mutant clones in plasma has been more recently reported in some patients with primary RAS mutant cancers, supporting for the first time an unexpected negative selection of RAS mutations during the clonal evolution of mCRC. To date, the extent of conversion to RAS wild type disease at the time of progression has not been clarified yet. As a proof of concept, we prospectively enrolled mCRC patients progressing under anti-VEGF based treatments. Idylla™ system was used to screen RAS mutations in plasma and the wild type status of RAS was further confirmed through IT-PGM (Ion Torrent Personal Genome Machine) sequencing. RAS was found mutant in 55% of cases, retaining the same plasma mutation as in the primary tumor at diagnosis, while it was found wild-type in 45%. Four patients testing negative for RAS mutations in plasma at the time of progression of disease (PD) were considered eligible for treatment with EGFR inhibitors and treated accordingly, achieving a clinical benefit. We here propose a hypothetical algorithm that accounts for the transient disappearance of RAS mutant clones over time, which might extend the continuum of care of mutant RAS colorectal cancer patients through the delivery of a further line of therapy. Full article

Quick and accurate molecular testing is necessary for the better management of many inherited diseases. Recent technological advances in various next generation sequencing (NGS) platforms, such as target panel-based sequencing, has enabled comprehensive, quick, and precise interrogation of many genetic variations. As a result, these technologies have become a valuable tool for gene discovery and for clinical diagnostics. The AmpliSeq Inherited Disease Panel (IDP) consists of 328 genes underlying more than 700 inherited diseases. Here, we aimed to assess the performance of the IDP as a sensitive and rapid comprehensive gene panel testing. A total of 88 patients with inherited diseases and causal mutations that were previously identified by Sanger sequencing were randomly selected for assessing the performance of the IDP. The IDP successfully detected 93.1% of the mutations in our validation cohort, achieving high overall gene coverage (98%). The sensitivity for detecting single nucleotide variants (SNVs) and short Indels was 97.3% and 69.2%, respectively. IDP, when coupled with Ion Torrent Personal Genome Machine (PGM), delivers comprehensive and rapid sequencing for genes that are responsible for various inherited diseases. Our validation results suggest the suitability of this panel for use as a first-line screening test after applying the necessary clinical validation. Full article

The Ion Torrent Personal Genome Machine (Ion PGM) is a semiconductor-based sequencing technology that is high quality, scalable, and economic. Its applications include genomic sequencing, drug resistance testing, microbial characterization, and targeted sequencing in cancer studies. However, little is known about the application of Ion PGM in cutaneous squamous cell carcinoma (cSCC). We therefore investigated the utility and validity of Ion PGM in cSCC and also gained a better understanding of the underlying molecular biology of cSCC. We detected novel gene mutations (KDR, FGFR2, and EGFR) in two cSCC patients. Moreover, we validated these mutations by pyrosequencing and Sanger sequencing. Our results indicated that the mutation screen using Ion PGM is consistent with traditional sequencing methods. Notably, these identified mutations were present at significantly higher rates in high-risk cSCC. Our results demonstrate a method to detect targetable genes in high-risk cSCC, and suggest that Ion PGM may enable therapeutic decision-making and future potential targets for personalized therapies in cSCC. Full article

Application of next-generation sequencing (NGS) technology to routine clinical practice has enabled characterization of personalized cancer genomes to identify patients likely to have a response to targeted therapy. The proper selection of tumor sample for downstream NGS based mutational analysis is critical to generate accurate results and to guide therapeutic intervention. However, multiple pre-analytic factors come into play in determining the success of NGS testing. In this review, we discuss pre-analytic requirements for AmpliSeq PCR-based sequencing using Ion Torrent Personal Genome Machine (PGM) (Life Technologies), a NGS sequencing platform that is often used by clinical laboratories for sequencing solid tumors because of its low input DNA requirement from formalin fixed and paraffin embedded tissue. The success of NGS mutational analysis is affected not only by the input DNA quantity but also by several other factors, including the specimen type, the DNA quality, and the tumor cellularity. Here, we review tissue requirements for solid tumor NGS based mutational analysis, including procedure types, tissue types, tumor volume and fraction, decalcification, and treatment effects. Full article