Rapid detection of mutations in CSF-cfTNA with the Genexus Integrated Sequencer.

PURPOSE: Genomic alterations are fundamental for molecular-guided therapy in patients with breast and lung cancer. However, the turn-around time of standard next-generation sequencing assays is a limiting factor in the timely delivery of genomic information for clinical decision-making. METHODS: In this study, we evaluated genomic alterations in 54 cerebrospinal fluid samples from 33 patients with metastatic lung cancer and metastatic breast cancer to the brain using the Oncomine Precision Assay on the Genexus sequencer. There were nine patients with samples collected at multiple time points. RESULTS: Cell-free total nucleic acids (cfTNA) were extracted from CSF (0.1-11.2 ng/µl). Median base coverage was 31,963× with cfDNA input ranging from 2 to 20 ng. Mutations were detected in 30/54 CSF samples. Nineteen (19/24) samples with no mutations detected had suboptimal DNA input (< 20 ng). The EGFR exon-19 deletion and PIK3CA mutations were detected in two patients with increasing mutant allele fraction over time, highlighting the potential of CSF-cfTNA analysis for monitoring patients. Moreover, the EGFR T790M mutation was detected in one patient with prior EGFR inhibitor treatment. Additionally, ESR1 D538G and ESR1::CCDC170 alterations, associated with endocrine therapy resistance, were detected in 2 mBC patients. The average TAT from cfTNA-to-results was < 24 h. CONCLUSION: In summary, our results indicate that CSF-cfTNA analysis with the Genexus-OPA can provide clinically relevant information in patients with brain metastases with short TAT.

Accurate Detection of SARS-CoV-2 by Next-Generation Sequencing in Low Viral Load Specimens.

As new SARS-CoV-2 variants emerge, there is an urgent need to increase the efficiency and availability of viral genome sequencing, notably to detect the lineage in samples with a low viral load. SARS-CoV-2 genome next-generation sequencing (NGS) was performed retrospectively in a single center on 175 positive samples from individuals. An automated workflow used the Ion AmpliSeq SARS-CoV-2 Insight Research Assay on the Genexus Sequencer. All samples were collected in the metropolitan area of the city of Nice (France) over a period of 32 weeks (from 19 July 2021 to 11 February 2022). In total, 76% of cases were identified with a low viral load (Ct ≥ 32, and ≤200 copies/µL). The NGS analysis was successful in 91% of cases, among which 57% of cases harbored the Delta variant, and 34% the Omicron BA.1.1 variant. Only 9% of cases had unreadable sequences. There was no significant difference in the viral load in patients infected with the Omicron variant compared to the Delta variant (Ct values, p = 0.0507; copy number, p = 0.252). We show that the NGS analysis of the SARS-CoV-2 genome provides reliable detection of the Delta and Omicron SARS-CoV-2 variants in low viral load samples.

Genetic Profiling of Non-Small Cell Lung Cancer in Moroccan Patients by Targeted Next-Generation Sequencing.

OBJECTIVES: We retrospectively analyzed the next-generation sequencing (NGS) results from diagnosed NSCLC patients to identify and compare genomic alterations of NSCLC between Moroccan patients and the Cancer Genome Atlas (TCGA). We also aimed to investigate the distribution and frequency of concurrent genomic alterations. METHODS: From December 2022 to December 2023, a retrospective study of 76 formalin-fixed paraffin-embedded (FFPE) samples have been profiled using the Oncomine™ Precision Assay on the Ion Torrent™ Genexus™ Integrated Sequencer across the panel of 50 key genes that are applicable for the selection of targeted therapy. RESULTS: Seventy of the 76 FFPE sequenced samples carried at least one genetic alteration in the tested genes. The study identified 234 genetic alterations in 18 genes. Targetable genetic alterations in EGFR, KRAS, MET, BRAF, ALK, RET and ROS1 were identified in 84.3% of tumors. EGFR and KRAS mutations were frequently reported, occurring in 24.3% and 22.9% of cases, respectively. The untargetable genetic alterations were found in 74.3% of the specimens in FGFR3, TP53, ERBB2, PIK3CA, CDKN2A, PDL1, FGFR1, PTEN, CHEK2 and ERBB3. There were additional uncommon/rare mutations in EGFR, BRAF, RET and ROS1. Comparing the prevalence of selected mutated genes in the NSCLC patients from the TCGA database identified substantial differences in EGFR (24.3%, vs14.97%), KRAS (22.9%, vs 25.99%), and TP53 (34.3%, vs 50.94%). ALK, ROS1, and RET gene rearrangements were detected in 4.3% of the 70 tumors tested. The ALK/RET/MET/ROS1/EML4 fusions were detected in 11.4% of samples. Co-alterations occurred in 67.1% of specimens. Co-occurring driver gene mutations were observed in 44.3%. TP53 mutations co-occurred driver gene mutations in 30% of tumors. Three cases (4.3%) harbored concurrent FGFR3, TP53, and PIK3CA alterations. CONCLUSION: Our results regarding the proportion of samples with actionable mutations demonstrate the value of NGS testing for NSCLC patients in a real-world clinical diagnostic setting.

Detection of Mycobacterium bovis in nasal swabs from communal goats (Capra hircus) in rural KwaZulu-Natal, South Africa.

Animal tuberculosis, caused by Mycobacterium bovis, presents a significant threat to both livestock industries and public health. Mycobacterium bovis tests rely on detecting antigen specific immune responses, which can be influenced by exposure to non-tuberculous mycobacteria, test technique, and duration and severity of infection. Despite advancements in direct M. bovis detection, mycobacterial culture remains the primary diagnostic standard. Recent efforts have explored culture-independent PCR-based methods for identifying mycobacterial DNA in respiratory samples. This study aimed to detect M. bovis in nasal swabs from goats (Capra hircus) cohabiting with M. bovis-infected cattle in KwaZulu-Natal, South Africa. Nasal swabs were collected from 137 communal goats exposed to M. bovis-positive cattle and 20 goats from a commercial dairy herd without M. bovis history. Swabs were divided into three aliquots for analysis. The first underwent GeneXpert® MTB/RIF Ultra assay (Ultra) screening. DNA from the second underwent mycobacterial genus-specific PCR and Sanger sequencing, while the third underwent mycobacterial culture followed by PCR and sequencing. Deep sequencing identified M. bovis DNA in selected Ultra-positive swabs, confirmed by region-of-difference (RD) PCR. Despite no other evidence of M. bovis infection, viable M. bovis was cultured from three communal goat swabs, confirmed by PCR and sequencing. Deep sequencing of DNA directly from swabs identified M. bovis in the same culture-positive swabs and eight additional communal goats. No M. bovis was found in commercial dairy goats, but various NTM species were detected. This highlights the risk of M. bovis exposure or infection in goats sharing pastures with infected cattle. Rapid Ultra screening shows promise for selecting goats for further M. bovis testing. These techniques may enhance M. bovis detection in paucibacillary samples and serve as valuable research tools.

Ion Torrent ™ Genexus ™ Integrated Sequencer and ForeNGS Analysis Software-An automatic NGS-STR workflow from DNA to profile for forensic science.

The Ion Torrent ™ Genexus ™ Sequencer (Genexus) is a highly integrated instrument that can automate library construction, templating, and sequencing in a single-instrument run. By programing the ForeNGS Analysis Software (FNAS), we bridged the gap between sequencing and genotyping without manual intervention. FNAS can automatically transfer sequencing output files from Genexus, analyze the repeat and flanking regions aligned to the GRCh38 assembly, name the alleles according to the ISFG guidelines, and generate user-friendly interactive profiles. Genexus and FNAS can accomplish the fully automatic DNA-to-Profile workflow in forensics. Based on our experiences, the optimal assay parameters on Genexus were validated as follows: 24 cycles of target amplification for library construction; 40 µL of library and 400 bp of template size for templating; 852 flows of dNTPs by order of Ion samba HID2 for sequencing; and 750,000 reads per sample at minimum for 16 samples multiplexed on a lane. By developmental validations of the Precision ID Globalfiler ™ NGS STR Panel v2, Genexus presented competitive performance at the optimal assay parameters qualified to detect commonly used forensic STR markers. It could produce repeatable and reproducible results, and human profiles could be easily separated from nonhuman profiles. Additionally, Genexus was sensitive enough to detect samples with 100 pg of input DNA, and it was suitable for various types of case samples, especially for low copy number samples and degraded samples. Moreover, minor contributors could be detected between the 4:1 and 1:4 mixtures with an analysis threshold of 50 × . The Genexus workflow is a robust and labor-effective solution enabling forensic scientists to obtain NGS-STR profiles within a single day and with only the need to prepare DNA extracts, then set up Genexus, and finally interpret profiles on FNAS.

Setting-Up a Rapid SARS-CoV-2 Genome Assessment by Next-Generation Sequencing in an Academic Hospital Center (LPCE, Louis Pasteur Hospital, Nice, France).

Introduction: Aside from the reverse transcription-PCR tests for the diagnosis of the COVID-19 in routine clinical care and population-scale screening, there is an urgent need to increase the number and the efficiency for full viral genome sequencing to detect the variants of SARS-CoV-2. SARS-CoV-2 variants assessment should be easily, rapidly, and routinely available in any academic hospital. Materials and Methods: SARS-CoV-2 full genome sequencing was performed retrospectively in a single laboratory (LPCE, Louis Pasteur Hospital, Nice, France) in 103 SARS-CoV-2 positive individuals. An automated workflow used the Ion Ampliseq SARS-CoV-2 panel on the Genexus Sequencer. The analyses were made from nasopharyngeal swab (NSP) (n = 64) and/or saliva (n = 39) samples. All samples were collected in the metropolitan area of the Nice city (France) from September 2020 to March 2021. Results: The mean turnaround time between RNA extraction and result reports was 30 h for each run of 15 samples. A strong correlation was noted for the results obtained between NSP and saliva paired samples, regardless of low viral load and high (>28) Ct values. After repeated sequencing runs, complete failure of obtaining a valid sequencing result was observed in 4% of samples. Besides the European strain (B.1.160), various variants were identified, including one variant of concern (B.1.1.7), and different variants under monitoring. Discussion: Our data highlight the current feasibility of developing the SARS-CoV-2 next-generation sequencing approach in a single hospital center. Moreover, these data showed that using the Ion Ampliseq SARS-CoV-2 Assay, the SARS-CoV-2 genome sequencing is rapid and efficient not only in NSP but also in saliva samples with a low viral load. The advantages and limitations of this setup are discussed.