Ion AmpliSeq Research Articles

Evaluation of the Thermo Fisher Oncomine Myeloid MRD DNA Assay on the Ion Torrent Platform

Introduction Malignant myeloid diseases are a group of blood cancers that arise from genetic changes in hematopoietic cells in the bone marrow or peripheral blood. The presence of measurable residual disease (MRD), or detection of abnormal cells in the body following completion of therapy, is associated with a higher risk of disease relapse, and decreased overall survival (Schuurhuis 2018). Next-generation sequencing (NGS) can be leveraged as a high-sensitivity, minimally invasive method for detection of MRD. The Ion Torrent Oncomine Myeloid MRD Assay (Myeloid MRD) is a targeted NGS assay designed for the detection of single nucleotide variants (SNVs) and insertions/deletions in a curated selection of clinically relevant myeloid disease-related genes at low limits of detection. Assay performance was evaluated using normal and diseased clinical blood and bone marrow samples and NGS reference materials. Methods The Myeloid MRD DNA panel targets hotspot regions across 33 genes and uses Ion AmpliSeq HD technology with unique molecular tagging to suppress sequencing artifacts and improve analytical sensitivity. Briefly, DNA was extracted from whole blood or bone marrow samples and 120 ng of nucleic acid was used as input for the assay. The DNA was divided between 2 polymerase chain reactions (PCR), where it served as the template for target amplifications, before combining reaction products by sample. Library preparation followed the standard Ion AmpliSeq HD protocol, where amplicons were partially enzymatically digested, and a second round of amplification was performed using barcoded primers. Following bead purification, the final library was measured by quantitative PCR (qPCR) to inform final library loading onto the sequencer. Six libraries were pooled and sequenced on an Ion 550 chip using the Ion GeneStudio S5 System. Primary and secondary analyses were performed using the Torrent Suite software, and results were exported to the Ion Reporter software for variant calling. From the variant calling results, a probit model was utilized to determine the variant allele frequency (VAF) at which the assay achieved 90% power to call an expected variant, defining the limit of detection (LoD). Other parameters evaluated included analytical sensitivity, analytical specificity, positive predictive value (PPV), accuracy, and precision. Results Overall, consistent, high quality on-target reads were observed across 22 sequencing runs with a median Ion Sphere Particle (ISP) loading of 90%, usable sequence of 59%, and over 130 million total reads per chip. When evaluating individual samples according to the vendor-recommended number of mapped reads (> 10 million), 98.3% of samples tested passed quality control (QC). Additional sample QC metrics established during assay validation included thresholds of > 80% reads on-target, > 80% uniformity of base coverage, and a mean read length of at least 80 base pairs. The estimated LoD for the assay was 0.1% VAF for small variants (SNVs, insertions/deletions).Above the LoD, the assay demonstrated 96.03% sensitivity, 99.89% accuracy of base-calling, 100% specificity, and 98.83% PPV.Additionally,a comparison was performed usingmatched whole blood and bone marrow samples collected from patients with acute myeloid leukemia (AML) to evaluate the concordance of variant calling. It was determined that the same variants were identified across both sample types, although at higher allele frequency in bone marrow compared to whole blood. Conclusion This evaluation of the Myeloid MRD assay demonstrates that it is a precise and accurate assay with sensitivity that is acceptable for MRD analysis. Notably, variant detection was concordant between whole blood and bone marrow sample types collected from AML patients, with bone marrow providing improved sensitivity for low-frequency variant detection. The user-friendly workflow and integrated informatics pipeline offer opportunities for streamlined testing and interpretation of results. The Ion Torrent Myeloid MRD DNA assay is a powerful new tool for MRD detection across a growing myeloid disease target landscape.

B-240 Development of AcroMetrixTM Human Microbiome Quality Control Materials

Abstract Background The human microbiome is receiving significant attention given its critical roles in the maintenance of human health. The compositional and functional alterations of the human microbiota have been associated with various pathological conditions including colorectal cancer (CRC). As the number of human metagenomic data increases, the interest to utilize these metagenomic biomarkers for diagnosis of colorectal cancer and other diseases has also elevated. Studies that evaluated the defined fecal microbial markers for CRC detection showed the test accuracy could vary from 73% to 98% depending on the detection panels of microbiome biomarkers. The measurement inconsistencies between laboratories due to the use of different detection platforms, the coverage of various assay panels, the biases in different steps of microbiome workflow all indicated the critical need for reliable quality control materials. Currently, the commercially available microbial standards usually contain limited species and also mixed with fixed ratios which may not reflect the microbial composition in the clinical samples. There is no commutable reference material that recapitulates the dysbiotic microbial composition under healthy or pathological conditions (e.g., CRC). Therefore, in this study, we developed DNA quality control materials (QCMs) that recapitulate the dysbiotic microbial composition for colorectal cancer to assess the technical and non-technical variation for part of the microbiome workflow. Methods We first identified the specific bacterial species that either are enriched in CRC patients or healthy human gut based on comprehensive literature search and the other available dataset. We then created microbiome QCMs to recapitulate their dysbiotic microbial composition in comparison to the compositions in healthy condition. The QCM consists of a mixture of genomic DNA from the selected bacterial species, the copy number of DNA was determined by Bio-RadTM droplet digital PCR (ddPCR) with the assays that target to species-specific regions to reflect the microbial composition. In addition, the microbiome controls are further evaluated and validated by Microbiome Health Research Assay on Ion Ampliseq and GeneStudio S5 platforms. Results The AcroMetrixTM human microbiome QCMs have been carefully formulated to mimic dysbiotic microbial composition for colorectal cancer or healthy human gut. This CRC QCM covers 29 different species with a wide range of guanine-cytosine content sequences, belonging to 10 phyla and 20 genera. 25 out of these 29 species (86.2%) were covered by Microbiome Health Research Assay. To mimic the trends of dysbiotic microbial composition, the DNA frequency among these 29 species was targeted from 0% to 15% according to the microbial composition. Droplet digital PCR results confirmed the target frequencies within the accepted tolerance range and the results were compared with the data from the Next Generation Sequencing platform. Conclusions The human microbiome QCMs that cover 29 bacterial species and mimic dysbiotic microbial composition for colorectal cancer or healthy human gut have been designed and developed. These QCMs could provide essential elements to verify, validate and monitor test performance, technical and non-technical variation of microbiome workflows, as well as product QC releases and method development for ddPCR & NGS assays.

Introduction of a multiplex amplicon sequencing assay to quantify DNA methylation in target cytosine markers underlying four selected epigenetic clocks

BackgroundDNA methylation analysis has proven to be a powerful tool for age assessment. However, the implementation of epigenetic age prediction in diagnostics or routine forensic casework requires appropriate laboratory methods. In this study, we aimed to compare the performance of large-scale DNA methylation analysis protocols that show promise in terms of accuracy, throughput, multiplexing capacity, and high sensitivity.ResultsThe protocols were designed to target a predefined panel of 161 genomic CG/CA sites from four known estimators of epigenetic age-related parameters, optimized and validated using artificially methylated controls or blood samples. We successfully targeted 96% of these loci using two enrichment protocols: Ion AmpliSeq™, an amplicon-based method integrated with Ion Torrent S5, and SureSelectXT Methyl-Seq, a hybridization-based method followed by MiSeq FGx sequencing. Both protocols demonstrated high accuracy and robustness. Although hybridization assays have greater multiplexing capabilities, the best overall performance was observed for the amplicon-based protocol with the lowest variability in DNA methylation at 25 ng of starting DNA, mean observed marker coverage of ~ 6.7 k reads, and accuracy of methylation quantification with a mean absolute difference between observed and expected methylation beta value of 0.054. The Ion AmpliSeq method correlated strongly with genome-scale EPIC microarray data (R = 0.91) and showed superiority in terms of methylation measurement accuracy. Method-to-method bias was accounted for by the use of linear transformation, which provided a highly accurate prediction of calendar age with a mean absolute error of less than 5 years for the VISAGE and Hannum age clocks used. The pace of aging (PoAm) and the mortality risk score (MRS) estimators included in our panel represent next-generation clocks, were found to have low to moderate correlations with the VISAGE and Hannum models (R < 0.75), and thus may capture different aspects of epigenetic aging.ConclusionsWe propose a laboratory tool that allows the quantification of DNA methylation in cytosines underlying four different clocks, thus providing broad information on epigenetic aging while maintaining a reasonable number of CpG markers, opening the way to a wide range of applications in forensics, medicine, and healthcare.

Genotypic variability in patients with clinical diagnosis of Bartter syndrome type 3

Bartter syndrome (BS) is a salt-losing hereditary tubulopathy characterized by hypokalemic metabolic alkalosis with secondary hyperaldosteronism. Confirmatory molecular diagnosis may be difficult due to genetic heterogeneity and overlapping of clinical symptoms. The aim of our study was to describe the different molecular findings in patients with a clinical diagnosis of classic BS. We included 27 patients (26 families) with no identified pathogenic variants in CLCNKB. We used a customized Ion AmpliSeq Next-Generation Sequencing panel including 44 genes related to renal tubulopathies. We detected pathogenic or likely pathogenic variants in 12 patients (44%), reaching a conclusive genetic diagnosis. Variants in SLC12A3 were found in 6 (Gitelman syndrome). Median age at diagnosis was 14.6 years (range 0.1–31), with no history of prematurity or polyhydramnios. Serum magnesium level was low in 2 patients (33%) but urinary calcium excretion was normal or low in all, with no nephrocalcinosis. Variants in SLC12A1 were found in 3 (BS type 1); and in KCNJ1 in 1 (BS type 2). These patients had a history of polyhydramnios in 3 (75%), and the mean gestational age was 34.2 weeks (SD 1.7). The median age at diagnosis was 1.8 years (range 0.1–6). Chronic kidney disease and nephrocalcinosis were present in 1 (25%) and 3 (75%) patients, respectively. A variant in CLCN5 was found in one patient (Dent disease), and in NR3C2 in another patient (Geller syndrome). Genetic diagnosis of BS is heterogeneous as different tubulopathies can present with a similar clinical picture. The use of gene panels in these diseases becomes more efficient than the study gene by gene with Sanger sequencing.

420 Molecular assessment of atopic dermatitis and psoriasis samples collected using a noninvasive technique

Abstract Updates in the molecular understanding of common and often debilitating skin diseases such as atopic dermatitis (AD) and psoriasis led to the development of multiple targeted systemic drugs. Optimal response to these medications relies on correct diagnosis, as well as clinical, personal and molecular factors unique to each patient. Currently, unstandardized assessment of clinical characteristics and comorbidities drive the development of a therapeutic plan for patients with AD and psoriasis. The molecular mechanisms underlying each patient’s disease are not routinely assessed when developing a treatment plan. This empirical approach to treatment selection delays optimal therapeutic response and leads to increased costs to healthcare systems. Therefore, incorporating molecular information from an individual patient’s disease should inform better treatment decisions. Previously, we presented the proof of concept for a noninvasive skin scraping sample collection technique by assessing gene expression differences of select genes of interest from psoriasis and AD samples using quantitative polymerase chain reaction. Indeed, candidate genes were differentially expressed in AD and psoriasis lesions relative to nonlesional skin and in AD relative to psoriasis lesions, demonstrating the feasibility of this collection technique. An algorithm based on the gene expression profiles of AD and psoriasis samples could be used to help guide therapy selection. This study aims to assess the molecular profiles of AD and psoriasis samples collected using a noninvasive skin scraping method with the ultimate goal of developing and validating a molecular test for these common inflammatory skin diseases. The superficial epidermis of lesional and nonlesional skin from patients with AD or psoriasis from three dermatology centers in the USA was collected by gently scraping the skin 10 times with a curette and immediately preserving the sample in a proprietary buffer. RNA isolated from samples was stored at −80°C until further use. Library preparation and next generation RNA sequencing was performed using the Ion AmpliSeq Transcriptome Human Gene Expression panel on the S5XL sequencer (ThermoFisher). Differential expression of genes was assessed using DESeq2; Metascape was used to assess enriched gene ontology of differentially expressed genes. When comparing AD lesional and nonlesional skin, 1633 transcripts were differentially expressed (absolute value of log2fold change &amp;gt;1.0 and padj &amp;lt; 0.05). Additionally, 4468 transcripts were differentially expressed between psoriasis lesional and nonlesional skin. Further, principal component analysis demonstrated that lesional and nonlesional samples for both AD and psoriasis could be distinguished by their respective gene expression profiles. Gene ontology enrichment analysis revealed that innate and adaptive immune system and cytokine signaling genes were among the most common types of differentially expressed genes between lesional and nonlesional samples. Moreover, gene expression differed between lesional AD and psoriasis samples. These results suggest that a molecular test could be developed from AD and psoriasis samples collected by a noninvasive scraping technique. Further, clinical correlation with therapeutic outcomes may be used in conjunction with molecular profiles to develop an algorithm to predict therapeutic response in these common inflammatory skin diseases.

Differential Gene Expression Signatures and Cellular Signaling Pathways induced by Lamin A/C Transcript Variants in MCF7 Cell Line.

Lamins are the major component of nuclear lamina. Alternative splicing of the 12 exons comprising lamin A/C gene creates five known transcript variants, lamin A, lamin C, lamin AΔ10, lamin AΔ50, and lamin C2. The main objective for this study was to examine the association of critical pathways, networks, molecular and cellular functions regulated by each Lamin A/C transcript variants. Ion AmpliSeq Transcriptome Human Gene Expression analysis was performed on MCF7 cells stably transfected with lamin A/C transcript variants. Lamin A or lamin AΔ50 upregulation was associated with activation of cell death and inactivation of carcinogenesis while both lamin C or lamin AΔ10 upregulation activated carcinogenesis and cell death. Data suggest anti-apoptotic and anti-senescence effects of lamin C and lamin AΔ10 as several functions, including apoptosis and necrosis functions are inactivated following lamin C or lamin AΔ10 upregulation. However, lamin AΔ10 upregulation is associated with a more carcinogenic and aggressive tumor phenotype. Lamin A or lamin AΔ50 upregulation is associated with a predicted activation of increased cell death and inactivation of carcinogenesis. Thus, different signaling pathways, networks, molecular and cellular functions are activated/inactivated by lamin A/C transcript variants resulting in a large number of laminopathies.

Massively parallel sequencing of 74 microhaplotypes and forensic characteristics in three Chinese Sino-Tibetan populations

Microhaplotype (MH), as an emerging type of forensic genetic marker in recent years, has the potential to support multiple forensic applications, especially for mixture deconvolution and biogeographic ancestry inference. Herein, we investigated the genotype data of 74 MHs included in a novel MH panel, the Ion AmpliSeq MH-74 Plex Microhaplotype Research Panel, in three Chinese Sino-Tibetan populations (Han, Tibetan, and Yi) using the Ion Torrent semiconductor sequencing. The sequencing performance, allele frequencies, effective number of alleles (Ae), informativeness (In), and forensic parameters were subsequently estimated and calculated. In addition, principal component analysis (PCA) and structure analysis were performed to explore the population relationships among the three populations and the ancestry component distribution. Overall, this novel MH panel is robust and reliable, and has an excellent sequencing performance. The Ae values ranged from 1.0126 to 7.0855 across all samples, and 75.68 % of MHs had Ae values >2.0000. Allele frequencies at some loci varied considerably among the three studied populations, and the mean In value was 0.0195. Moreover, the genetic affinity between Tibetans and Yis was closer than that between Tibetans and Hans. The aforementioned results suggest that the Ion AmpliSeq MH-74 Plex Microhaplotype Research Panel is highly polymorphic in three investigated populations and could be used as an effective tool for human forensics. Although these 74 MHs have demonstrated the competency in continental population stratification, a higher resolution for distinguishing intracontinental subpopulations and a more comprehensive database with sufficient reference population data still remain to be accomplished.

Phase II randomized window of opportunity trial evaluating cytotoxic and immunomodulatory effects of intratumoral INT230-6 in early stage breast cancer: The INVINCIBLE trial.

573 Background: Most breast cancers are considered immunological cold and are therefore minimally responsive to immunotherapies. Local cytotoxic therapies can induce cell death, expose tumor antigens, provide adjuvants for anti-tumor immune priming, and potentially increase responsiveness to immunotherapies. We conducted a randomized, double blind, Phase 2 presurgical Window-Of-Opportunity trial for intratumoral (IT) INT230-6 (comprising VINblastine (VIN) Cisplatin (VIN)) evaluating clinical and BioLogical Effects in patients with early-stage operable Breast Cancer (the INVINCIBLE trialNCT04781725. The trial evaluated the immune response within the tumor and microenvironment in vivo after IT INT230-6 injections. Methods: Women with newly diagnosed and awaiting surgery for early-stage intermediate or high-grade ER+ T1-T2 invasive breast cancers were recruited to the trial. Participating patients were allocated (2:1) to IT injections of INT230-6 vs saline placebo, prior to resection. RNA profiling of the pre- (baseline biopsy) and post- treatment (surgical resection) tumours using the Ion AmpliSeq Transcriptome Human Gene Expression (Thermo Fisher Scientific) and DNA sequencing using a large (500 gene) comprehensive genomic profiling panel (OCAPlus,) was performed. Results: Significant tumor necrosis was observed in the majority of subjects injected with INT230-6. In the 60 pre-treatment biopsies profiled with OCAPlus, the genes most frequently mutated included PIK3CA (38%), TP53 (23%), CDH1 (22%) and TBX3 (17%). Frequent copy number changes were identified in CCND1 (23%), FGFR1 (18%), and CDH1 (17%). Gene expression analysis showed significant differential gene expression between the baseline biopsy and surgical specimens in patients treated with the drug. Pathway analysis identified genes associated with TCR signaling, macrophage markers, IL-18 signaling and B cell receptor signaling were significantly up-regulated in the post treatment samples. Conclusions: This is the first study evaluating the potential biologic effects of intratumoral cytotoxicity with INT230-6 and its role as a potential immune priming therapy in traditional immune quiescent ER+ breast cancers.

Identification of Genomic Alterations in Thai Patients With Colorectal Cancer Using Next-Generation Sequencing-Based Multigene Cancer Panel.

Introduction Colorectal cancer (CRC) is one of the leading causes of death and illness in the general population. Although the incidence of CRC is steadily decreasing worldwide, it is being diagnosed more in individuals under 50 years of age. Multiple disease-causing variants have been reported to be involved in the development of CRC. This study aimed to investigate the molecular and clinical characteristics of Thai patients with CRC. Methods NGS-based multigene cancer panel testing was performed on 21 unrelated patients. Target enrichment was performed using a custom-designed Ion AmpliSeq on-demand panel. Thirty-six genes associated with CRC and other cancer were analyzed for variant detection. Results Sixteen variants (five nonsense, eight missense, two deletions, and one duplication) in nine genes were identified in 12 patients. Eight (66.7%) patients harboring disease-causing deleterious variants in genes APC, ATM, BRCA2, MSH2, and MUTYH. One of the eight patients also carried additional heterozygous variants in genes ATM, BMPR1A, and MUTYH. In addition, four patients carried variants of uncertain significance in genes APC, MLH1, MSH2, STK11, and TP53. Among all detected genes, APC was the most frequent causative gene observed in CRC patients, which is consistent with previous reports. Conclusion This study demonstrated the comprehensive molecular and clinical characterization of CRC patients. These findings showed the benefits of using multigene cancer panel sequencing for pathogenic gene detection and showed the prevalence of genetic aberrations in Thai patients with CRC.

Efficient SARS-CoV-2 Surveillance during the Pandemic-Endemic Transition Using PCR-Based Genotyping Assays.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VOC) pose an increased risk to public health due to higher transmissibility and/or immune escape. In this study, we assessed the performance of a custom TaqMan SARS-CoV-2 mutation panel consisting of 10 selected real-time PCR (RT-PCR) genotyping assays compared to whole-genome sequencing (WGS) for identification of 5 VOC circulating in The Netherlands. SARS-CoV-2 positive samples (N = 664), collected during routine PCR screening (15 ≤ CT ≤ 32) between May-July 2021 and December 2021-January 2022, were selected and analyzed using the RT-PCR genotyping assays. VOC lineage was determined based on the detected mutation profile. In parallel, all samples underwent WGS with the Ion AmpliSeq SARS-CoV-2 research panel. Among 664 SARS-CoV-2 positive samples, the RT-PCR genotyping assays classified 31.2% as Alpha (N = 207); 48.9% as Delta (N = 325); 19.4% as Omicron (N = 129), 0.3% as Beta (N = 2), and 1 sample as a non-VOC. Matching results were obtained using WGS in 100% of the samples. RT-PCR genotyping assays enable accurate detection of SARS-CoV-2 VOC. Furthermore, they are easily implementable, and the costs and turnaround time are significantly reduced compared to WGS. For this reason, a higher proportion of SARS-CoV-2 positive cases in the VOC surveillance testing can be included, while reserving valuable WGS resources for identification of new variants. Therefore, RT-PCR genotyping assays would be a powerful method to include in SARS-CoV-2 surveillance testing. IMPORTANCE The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genome changes constantly. It is estimated that there are thousands of variants of SARS-CoV-2 by now. Some of those variants, variants of concern (VOC), pose an increased risk to public health due to higher transmissibility and/or immune escape. Pathogen surveillance helps researchers, epidemiologists, and public health officials to monitor the evolution of infectious diseases agents, alert on the spread of pathogens, and develop counter measures like vaccines. The technique used for the pathogen surveillance is called sequence analysis which makes it possible to examine the building blocks of SARS-CoV-2. In this study, a new PCR method based on the detection of specific changes of those building blocks is presented. This method enables a fast, accurate and cheap determination of different SARS-CoV-2 VOC. Therefore, it would be a powerful method to include in SARS-CoV-2 surveillance testing.

Performance of Ultra-Rapid Idylla™ EGFR Mutation Test in Non-Small-Cell Lung Cancer and Its Potential at Clinical Molecular Screening.

The Idylla™ EGFR Mutation Test is an ultra-rapid single-gene test that detects epidermal growth factor receptor (EGFR) mutations using formalin-fixed paraffin-embedded specimens. Here, we compared the performance of the Idylla EGFR Mutation Test with the Cobas® EGFR Mutation Test v2. Surgically resected NSCLC specimens obtained at two Japanese institutions (N = 170) were examined. The Idylla EGFR Mutation Test and the Cobas EGFR Mutation Test v2 were performed independently and the results were compared. For discordant cases, the Ion AmpliSeq Colon and Lung Cancer Research Panel V2 was performed. After the exclusion of five inadequate/invalid samples, 165 cases were evaluated. EGFR mutation analysis revealed 52 were positive and 107 were negative for EGFR mutation in both assays (overall concordance rate: 96.4%). Analyses of the six discordant cases revealed that the Idylla EGFR Mutation Test was correct in four and the Cobas EGFR Mutation Test v2 was correct in two. In a trial calculation, the combination of the Idylla EGFR Mutation Test followed by a multi-gene panel test will reduce molecular screening expenses if applied to a cohort with EGFR mutation frequency >17.9%. We demonstrated the accuracy and potential clinical utility of the Idylla EGFR Mutation Test as a molecular screening platform in terms of turnaround time and molecular testing cost if applied to a cohort with a high EGFR mutation incidence (>17.9%).

Next Generation Sequencing (NGS) Target Approach for Undiagnosed Dysglycaemia.

Next-generation sequencing (NGS) has revolutionized the field of genomics and created new opportunities for basic research. We described the strategy for the NGS validation of the "dysglycaemia panel" composed by 44 genes related to glucose metabolism disorders (MODY, Wolfram syndrome) and familial renal glycosuria using Ion AmpliSeq technology combined with Ion-PGM. Anonymized DNA of 32 previously genotyped cases with 33 different variants were used to optimize the methodology. Standard protocol was used to generate the primer design, library, template preparation, and sequencing. Ion Reporter tool was used for data analysis. In all the runs, the mean coverage was over 200×. Twenty-nine out of thirty three variants (96.5%) were detected; four frameshift variants were missed. All point mutations were detected with high sensitivity. We identified three further variants of unknown significance in addition to pathogenic mutations previously identified by Sanger sequencing. The NGS panel allowed us to identify pathogenic variants in multiple genes in a short time. This could help to identify several defects in children and young adults that have to receive the genetic diagnosis necessary for optimal treatment. In order not to lose any pathogenic variants, Sanger sequencing is included in our analytical protocol to avoid missing frameshift variants.

A comparison of five Illumina, Ion Torrent, and nanopore sequencing technology-based approaches for whole genome sequencing of SARS-CoV-2

Rapid identification of the rise and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern remains critical for monitoring of the efficacy of diagnostics, therapeutics, vaccines, and control strategies. A wide range of SARS-CoV-2 next-generation sequencing (NGS) methods have been developed over the last years, but cross-sequence technology benchmarking studies have been scarce. In the current study, 26 clinical samples were sequenced using five protocols: AmpliSeq SARS-CoV-2 (Illumina), EasySeq RC-PCR SARS-CoV-2 (Illumina/NimaGen), Ion AmpliSeq SARS-CoV-2 (Thermo Fisher), custom primer sets (Oxford Nanopore Technologies (ONT)), and capture probe-based viral metagenomics (Roche/Illumina). Studied parameters included genome coverage, depth of coverage, amplicon distribution, and variant calling. The median SARS-CoV-2 genome coverage of samples with cycle threshold (Ct) values of 30 and lower ranged from 81.6 to 99.8% for, respectively, the ONT protocol and Illumina AmpliSeq protocol. Correlation of coverage with PCR Ct values varied per protocol. Amplicon distribution signatures differed across the methods, with peak differences of up to 4 log10 at disbalanced positions in samples with high viral loads (Ct values ≤ 23). Phylogenetic analyses of consensus sequences showed clustering independent of the workflow used. The proportion of SARS-CoV-2 reads in relation to background sequences, as a (cost-)efficiency metric, was the highest for the EasySeq protocol. The hands-on time was the lowest when using EasySeq and ONT protocols, with the latter additionally having the shortest sequence runtime. In conclusion, the studied protocols differed on a variety of the studied metrics. This study provides data that assist laboratories when selecting protocols for their specific setting.

Comparison between Three Different Techniques for the Detection of EGFR Mutations in Liquid Biopsies of Patients with Advanced Stage Lung Adenocarcinoma

Oncogenic mutations in the EGFR gene are targets of tyrosine kinase inhibitors (TKIs) in lung adenocarcinoma (LC) patients, and their search is mandatory to make decisions on treatment strategies. Liquid biopsy of circulating tumour DNA (ctDNA) is increasingly used to detect EGFR mutations, including main activating alterations (exon 19 deletions and exon 21 L858R mutation) and T790M mutation, which is the most common mechanism of acquired resistance to first- and second-generation TKIs. In this study, we prospectively compared three different techniques for EGFR mutation detection in liquid biopsies of such patients. Fifty-four ctDNA samples from 48 consecutive advanced LC patients treated with TKIs were tested for relevant EGFR mutations with Therascreen® EGFR Plasma RGQ-PCR Kit (Qiagen). Samples were subsequently tested with two different technologies, with the aim to compare the EGFR detection rates: real-time PCR based Idylla™ ctEGFR mutation assay (Biocartis) and next-generation sequencing (NGS) system with Ion AmpliSeq Cancer Hotspot panel (ThermoFisher). A high concordance rate for main druggable EGFR alterations was observed with the two real-time PCR-based assays, ranging from 100% for T790M mutation to 94% for L858R variant and 85% for exon 19 deletions. Conversely, lower concordance rates were found between real-time PCR approaches and the NGS method (L858R: 88%; exon19-dels: 74%; T790M: 37.5%). Our results evidenced an equivalent detection ability between PCR-based techniques for circulating EGFR mutations. The NGS assay allowed detection of a wider range of EGFR mutations but showed a poor ability to detect T790M.

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.

Constant p.L424H Mutation in GTF2I in Micronodular Thymomas With Lymphoid Stroma: Evidence Supporting Close Relationship With Type A and AB Thymomas

Micronodular thymoma with lymphoid stroma is a rare thymic neoplasm characterized by discrete nodules of epithelial tumor cells separated by abundant lymphoid stroma. The genetic features of micronodular thymoma with lymphoid stroma remain largely unexplored. Owing to the interference of abundant intratumoral, nonneoplastic lymphoid cells, a highly sensitive approach is necessary to study genetic changes in these tumors. In this study, we used a highly sensitive next-generation sequencing assay using the molecular barcoding Ion AmpliSeq HD technology to study the most commonly mutated genes in thymomas, including GTF2I, HRAS, NRAS, KRAS, and TP53. A total of 12 cases of micronodular thymomas with lymphoid stroma were tested, and 2 cases also had areas of type A thymoma in their tumor bed. Two micronodular thymic carcinomas with lymphoid stroma, a histological mimic of micronodular thymoma, were also included for comparison. Recurrent p.L424H mutations in GTF2I were found in all the cases of micronodular thymoma with lymphoid stroma but not in the cases of micronodular thymic carcinomas. In addition, 3 cases of micronodular thymoma with lymphoid stroma also had concomitant HRAS and/or KRAS mutations. Our study showed that p.L424H mutations in GTF2I is a constant genetic feature of micronodular thymoma with lymphoid stroma. This finding strongly suggests that micronodular thymoma with lymphoid stroma is closely related to type A and AB thymomas because they all share p.L424H mutations in GTF2I.

Detection of mutations and fusions in lung adenocarcinoma using ion torrent sequenc-ing technology

Introduction. Adenocarcinomas are the most common lung tumors and are often diagnosed in advanced stages when the tumor has a polyclonal form with a wide variety of genetic alterations and activated mutational processes. Comprehensive analysis of mutational status from FFPE tissue samples in such patients can provide a therapeutic perspective and contribute considerably to clinical decisions thereby increasing the overall survival rate. &#x0D; Material and methods. 22 genes were analyzed for mutations and 4 genes for fusion transcripts using two Ion AmpliSeq panels. DNA was isolated from paraffin-embedded tissue sections while RNA analysis was performed using two types of samples: paraffin blocks and fresh tumor tissue. Key variant detection and data analysis was performed using next platforms: Ion Reporter, ONCOMINE, R language. &#x0D; Results. The study of 30 tumor samples allowed the detection of 147 mutations and 4 fusions in 19 genes, and the therapeutically actionable variants were associated with different drugs clinically approved or in the phase of clinical trials. The most genetic variants were identified in the TP53, EGFR and NOTCH1 genes with a prevalence of over 50% in the TP53 gene, while all 4 detected fusions (one fusion per sample) represent the association of the ALK gene with other partners: EML4(13)-ALK(20) – present in 2 samples; EML4(6)-ALK(20); and an ALK fusion with an unknown partner gene. &#x0D; Conclusions. Analyzing the mutational status of tumor samples from patients with lung adenocarcinoma it has been ascertained the therapeutic utility of gene panel sequencing covering point mutations, INDELs and SNVs, as well as gene fusions, using FFPE tissue as primary material. This is valid for both targeted monotherapies and combined therapies.

The proliferation of atypical hepatocytes and CDT1 expression in noncancerous tissue are associated with the postoperative recurrence of hepatocellular carcinoma

Recently, we reported that extent of proliferation of atypical hepatocytes (atypical hepatocytes) was most important histological risk factor for development of hepatocellular carcinoma (HCC) from chronic hepatitis C or liver cirrhosis. Here, we aimed to clarify whether the atypical hepatocytes in noncancerous sections is also involved in postoperative recurrence. Furthermore, we investigated significant genes involved in the atypical hepatocytes. Association between the extent of atypical hepatocytes in noncancerous tissue and postoperative recurrence was validated in 356 patients with HCC. Next, we identified putative signature genes involved in extent of atypical hepatocytes. First, atypical hepatocytes or hepatocytes other than the atypical hepatocyte in noncancerous sections of 4 HCC patients were selectively collected by laser capture microdissection (LCM). Second, the gene expression profiles of the selected hepatocyte populations were compared using Ion AmpliSeq Transcriptome Human Gene Expression Kit (Thermo Fisher SCIENTIFIC, Waltham, MA, USA) analysis. Finally, we validated the mRNA expression of the extracted genes in noncancerous frozen liver tissue from 62 patients with HCC by RT-qPCR to identify the signature genes involved in both the extent of atypical hepatocytes and postoperative recurrence. Furthermore, the extent of atypical hepatocytes and CDT1 expression in noncancerous sections from 8 patients with HCC were also validated by selectively collecting samples using LCM. The extent of atypical hepatocytes was associated with postoperative recurrence. Of the genes that showed significant differences in expression levels between two populations, the expression of the chromatin licensing and DNA replication factor 1 (CDT1) gene was most strongly associated with the extent of atypical hepatocytes and was also associated with postoperative recurrence. Furthermore, CDT1-positive cells that exhibited stronger expression resembled those morphologically considered to be atypical hepatocytes. CDT1 and Ki-67 were colocalized in the nuclei of both hepatocytes and cancer cells. The hepatocytes in noncancerous livers were not uniform in each hepatocyte population, suggesting that the accumulation of genetic abnormalities was variable. We found that the strong degree of atypical hepatocytes and high CDT1 mRNA expression represent a high carcinogenic state of the liver. Thus, we consider the evaluation of degree of these could support the personalized medicine.

Development and Assessment of a Novel Whole-Gene-Based Targeted Next-Generation Sequencing Assay for Detecting the Susceptibility of Mycobacterium tuberculosis to 14 Drugs.

Targeted next-generation sequencing (tNGS) has emerged as an alternative method for detecting drug-resistant tuberculosis (DR-TB). To provide comprehensive drug susceptibility information and to address mutations missed by available commercial molecular diagnostics, we developed and evaluated a tNGS panel with 22 whole-gene targets using the Ion Torrent platform to predict drug resistance to 14 drugs, namely, rifampicin (RIF), isoniazid (INH), ethambutol (EMB), pyrazinamide (PZA), moxifloxacin (MFX), levofloxacin (LFX), amikacin (AMK), capreomycin (CM), kanamycin (KM), streptomycin (SM), bedaquiline (BDQ), clofazimine (CFZ), linezolid (LZD), and delamanid (DLM). We selected 50 and 35 Mycobacterium tuberculosis isolates with various DR profiles as the training set and the challenge set, respectively. Comparative variant analyses of the DR genes were performed using Sanger sequencing and whole-genome sequencing (WGS). Phenotypic drug susceptibility testing (pDST) results were used as gold standards. Regarding the limit of detection, the tNGS assay detected 2.9 to 3.8% minority variants in 4% mutant mixtures. The sensitivity and specificity of tNGS were 97.0% (95% confidence interval [CI] = 93.1 to 98.7%) and 99.1% (95% CI = 97.7 to 99.7%), respectively. The concordance of tNGS with pDST was 98.5% (95% CI = 97.2 to 99.2%), which was comparable to that of WGS (98.7%, 95% CI = 97.4 to 99.3%) and better than that of Sanger sequencing (96.9%, 95% CI = 95.3 to 98.0%). The agreement between tNGS and pDST was almost perfect for RIF, INH, EMB, MFX, LFX, AMK, CM, KM, SM, BDQ, and LZD (kappa value = 0.807 to 1.000) and substantial for PZA (kappa value = 0.791). Our customized novel whole-gene-based tNGS panel is highly consistent with pDST and WGS for comprehensive and accurate prediction of drug resistance in a strengthened and streamlined DR-TB laboratory program. IMPORTANCE We developed and validated a tNGS assay that was the first to target 22 whole genes instead of regions of drug resistance genes and comprehensively detected susceptibility to 14 anti-TB drugs, with great flexibility to include new or repurposed drugs. Notably, we demonstrated that our custom-designed Ion AmpliSeq TB research panel platform had high concordance with pDST and could significantly reduce turnaround time (by approximately 70%) to meet a clinically actionable time frame. Our tNGS assay is a promising DST solution for providing needed clinical information for precision medicine-guided therapies for DR-TB and allows the rollout of active pharmacovigilance.

Make it "SNPPY" - Updates to SRM 2391d: PCR-Based DNA Profiling Standard

Standard Reference Material (SRM) 2391d: PCR-Based DNA Profiling Standard was released to the forensic community in 2019. Next Generation Sequencing (NGS) was used as the primary method of certification, where certified values were assigned when a high coverage sequence string was available for a marker. Using NGS to assign values has allowed for additional marker sets beyond short tandem repeat (STR) loci, including single nucleotide polymorphisms (SNPs) and mitochondrial DNA (mtDNA) whole genome sequences, to be included in the Certificate of Analysis (COA). Since the 2019 release, several commercial NGS panels have become available including the Verogen ForenSeq mtDNA Control Region, mtDNA Whole Genome, MainstAY, and Kintelligence Kits. In addition, three community Ion AmpliSeq panels from Thermo Fisher (MH-74 Plex, VISAGE, and Y-SNP) are now available. While the mtDNA whole genome sequence for the components are already included and no new STR markers are introduced by MainstAY, the other recently released panels allow for the inclusion of > 11,000 additional SNPs (e.g., identity, ancestry, phenotype, kinship, and X- and Y-SNPs) and 74 microhaplotypes to the COA for SRM 2391d in an update completed by fall of 2022.