Next-generation Sequencing Assay For Detection Research Articles

Analytical Validation of an Automated Semiconductor-Based Next-Generation Sequencing Assay for Detection of DNA and RNA Alterations in Myeloid Neoplasms

Myeloid neoplasms are heterogeneous tumors derived from early hematopoietic progenitors. Most international guidelines, including the European LeukemiaNet 2022 update, recommend testing a comprehensive set of genes, most within a 3- to 5-day period for optimal treatment decisions. Next-generation sequencing gene panels are essential for identifying genetic alterations, risk stratification, and determining targeted therapies for myeloid malignancies. This study describes the analytical validation of the Oncomine Myeloid Assay GX v2 (Myeloid GX v2) in combination with the Ion Torrent Genexus System using commercial controls, 16 variant-negative samples, and 130 clinical samples of myeloid neoplasms. The Myeloid GX v2 panel detected single nucleotide variants (SNVs), insertions/deletions (indels) (allele frequency >5%), and gene fusions (minimum 11 fusion copies/μL) in synthetic controls with a sensitivity of 100%. Specificity for detection of SNVs, indels, or fusions in 16 variant-negative samples was 100%. Sensitivity for detection of SNVs, indels, and gene fusions in 130 clinical samples was 99%, 97%, and 100%, respectively. Overall precision was 100% for SNVs, 96% for indels, and 100% for fusions. The average turnaround time from nucleic acid extraction to results was 2 days. The Myeloid GX v2 panel is highly accurate and reproducible for the detection of SNVs, indels, and gene fusions in myeloid neoplasms. The ability to deliver clinically relevant results in a short time is key to providing personalized treatments.

A comprehensive HPV-STI NGS assay for detection of 29 HPV types and 14 non-HPV sexually transmitted infections

BackgroundSexually transmitted infections (STIs) are prevalent throughout the world and impose a significant burden on individual health and public health systems. Missed diagnosis and late treatment of STIs can lead to serious complications such as infertility and cervical cancer. Although sexually transmitted co-infections are common, most commercial assays target one or a few STIs. The HPV-STI ChapterDx Next Generation Sequencing (NGS) assay detects and quantifies 29 HPVs and 14 other STIs in a single-tube and single-step PCR reaction and can be applied to tens to thousands of samples in a single sequencing run.MethodsA cohort of 274 samples, previously analyzed by conventional cytology/histology and Roche cobas HPV Test, were analyzed by ChapterDx HPV-STI NGS assay for detection of 43 HPV and STI. A set of 43 synthetic control DNA fragments for 43 HPV and STI were developed to evaluate the limit of detection, specificity, and sensitivity of ChapterDx HPV-STI NGS assay.ResultsThe assay was evaluated in this study, and the limit of detection was 100% at 50 copies for all targets, and 100%, 96%, 88% at 20 copies for 34, 8, and 1 target, respectively. The performance of this assay has been compared to Roche cobas HPV test, showing an overall agreement of 97.5% for hr-HPV, and 98.5% for both, HPV16 and HPV18. The assay also detected all HPV-infected CIN2/3 with 100% agreement with Roche cobas HPV results. Moreover, several co-infections with non-HPV STIs, such as C. trachomatis, T. vaginalis, M. genitalium, and HSV2 were identified.ConclusionsThe ChapterDx HPV-STI NGS assay is a user-friendly, easy to automate and cost-efficient assay, which provides accurate and comprehensive results for a wide spectrum of HPVs and STIs.

The clinical utility of a custom-developed targeted next-generation sequencing assay for detection of mutations associated with Philadelphia-negative chronic myeloproliferative neoplasms: Two case examples with CALR exon 9 mutations

We have developed a highly targeted custom next generation sequencing-based test targeting JAK2 exons 12 and 14, CALR exon 9, and MPL exon 10, which are implicated in Philadelphia-negative chronic myeloproliferative neoplasms. The assay is capable of ultra-high sequencing depth and producing mutational detection sensitivities of 0.5% and below. We have validated the performance of the test through orthogonal testing and demonstrated a high degree of multiplexing with up to 50 samples in a single run. We show the clinical utility of this test through the description of a couple of cases with myeloproliferative disorders with Type-II and Type-II-like mutations in exon 9 of the CALR gene.

Abstract 3624: Development of a comprehensive and highly sensitive next-generation sequencing assay for detection of copy number variations

Abstract Background: As our knowledge of how DNA alterations can drive cancer progression increases, assays that can simultaneously detect multiple types of variants in a simple and cost-effective manner are becoming increasingly crucial. This holds true of copy number variations (CNVs), where evaluation of this type of variant is an important and necessary feature of any solid tumor profiling assay. Conventional methods for detecting CNVs such as immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and quantitative PCR (qPCR) are limited to detecting only one gene amplification at a time. This can be a significant drawback with FFPE samples, where the DNA is of low abundance and often heavily degraded, while the number of gene amplifications known to be important in cancer continues to grow. Additionally, different tumor types may express amplifications at different rates and expression may be heterogeneous within the tumor, potentially with irregular staining patterns - all of which illustrate the need for new approaches to CNV detection. Methods: Next-generation sequencing (NGS) offers the ability to assess variants in multiple genes using one sample. To that end, Illumina is developing a comprehensive, hybrid capture-based NGS assay targeting 170 key cancer genes for sequencing on the NextSeq1 platform. The assay consists of a DNA workflow for the identification of single-nucleotide variants (SNVs), small insertions and deletions (indels), as well as an RNA workflow for the identification of splice variants and gene fusions. In addition, using the DNA workflow, a novel analysis pipeline, and CNV caller, CNVs from 57 different genes can be simultaneously assessed all by sequencing of a single sample. Results: Here we present data on both cell lines and FFPE samples of SNVs and indels down to 5% allele fraction and CNVs down to ∼2-fold amplification, all from 40 ng of DNA. To demonstrate the accuracy and precision of our CNV detection method, we tested 7 samples for CNVs using orthogonal CNV detection methods. The Illumina NGS assay detected ERBB2 amplifications in 4 out of 7 samples. Of the 4 Illumina NGS positive samples, 3 samples were positive by FISH and all 4 were positive by droplet digital PCR (ddPCR) and the Illumina TruSight Tumor 15 panel. The 3 samples that were negative for ERBB2 amplifications by the Illumina NGS assay were also negative by both FISH and ddPCR. Within these samples we also found a previously unknown FGFR1 amplification. Conclusions: The novel Illumina NGS library preparation method is an innovative and useful tool to find multiple CNVs, along with other variant types, within a single sample. The assay can detect multiple CNVs within a single FFPE sample and identify previously uncharacterized CNVs that could be important in finding the correct treatment for a cancer patient. Citation Format: Chia-Ling Hsieh, Clare Zlatkov, Byron Luo, Chen Zhao, Kathryn Stephens, Han-Yu Chuang, Lisa Kelly, Katherine Chang, Rachel Liang, Jianli Cao, Scott Lang, Ashley Adams, Naseem Ajili, Laurel Ball, Glorianna Caves, Danny Chou, Katie Clark, Brian Crain, Anthony Daulo, Sarah Dumm, Ridwana Ekram, Yonmee Han, Anne Jager, Suzanne Johansen, Li Teng, Jenn Lococo, Jaime McLean, Juli Parks, Jason Rostron, Jennifer Sayne, Jennifer Silhavy, June Snedecor, Mckenzi Toh, Stephanie Tong, Elizabeth Upsall, Paulina Walichiewicz, Xiao Chen, Amanda Young, Ali Kuraishy, Karen Gutekunst, Matt Friedenberg, Charles Lin. Development of a comprehensive and highly sensitive next-generation sequencing assay for detection of copy number variations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3624.

Abstract 506: Post-surgical resection monitoring in early stage colorectal carcinoma patients using a circulating cell-free DNA assay with ultra-high accuracy and specificity

Abstract Analysis of cell-free circulating tumor DNA (ctDNA) by next-generation sequencing (NGS) allows non-invasive real-time profiling of actionable genomic alterations. Liquid biopsy provides an option for disease monitoring in early stage cancer patients post surgical resection, with a potential to aid in adjuvant decision making. However, to be applicable, tests must cover a broad enough genomic footprint to not require a priori knowledge of mutations, have high specificity, and sensitivity higher than conventional methods. NGS is necessary, since inactivating mutations are the most common alteration type in many common cancer types such as colorectal carcinomas (CRC). Here we present a highly efficient and specific NGS assay for detection of ctDNA in early stage cancer patients, capable of detecting single molecule variants across a 12 kb gene panel with an analytical sensitivity of >0.02% for single nucleotide variants (SNVs) and indels. This panel was applied to a clinical study involving 14 early stage (II/III) CRC patients with both pre- and post-op blood draws (up to 7 days post surgery). A subset (6 patients) also had tumor samples collected at the time of the surgical resection of the tumor. Overall, the detection rate of ctDNA in pre-op blood draws was 93%. In the post-op blood draws ctDNA was detectable in 43% of cases. The estimated average minor allele frequency (MAF) is 0.58% (± 0.82%) in pre-op, 0.18% (±0.21%) in post-op, and 40% (±18%) in tumor samples. When tumor tissue was available and used as a reference, the clinical sensitivity, specificity, and accuracy in pre-op blood samples were 83%, 99.995%, and 99.99%, respectively. SNVs with MAF as low as 0.04% were confirmed in tissue data. The clinical specificity of variants detected in post-op blood samples using pre-op samples as the reference is 99.996%. Cohort expansion to 50 patients and follow-up for clinical recurrence in both cohorts is ongoing. In conclusion, we have developed an assay with ultra-high accuracy and specificity, for the detection of ctDNA in early stage CRC patients that is capable of detecting alterations present in the tumor post-surgical resection. This technology allows for a promising non-invasive route for molecular monitoring of residual disease post surgery and for early detection of relapse compared to traditional methodologies. Citation Format: Stefanie A. Mortimer, Katharine Dilger, Stephen Fairclough, Diana Abdueva, Darya Chudova, Ankit Sarin, Jim Leng, Jeeyun Lee, Helmy Eltoukhy, AmirAli Talasaz. Post-surgical resection monitoring in early stage colorectal carcinoma patients using a circulating cell-free DNA assay with ultra-high accuracy and specificity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 506.

Targeted next-generation sequencing assay for detection of mutations in primary myopathies

Mutations in more than 100 different genes are known to cause hereditary primary myopathies. In patients with less distinct phenotypes several genes may have to be sequenced in order to make the correct diagnosis. The large number of possible candidate genes and overlapping phenotypes, as well as an enormous size of some of the genes such as DMD, TTN and NEB, constitute difficult challenges for molecular genetic diagnostics using conventional sequencing. Molecular characterization is nevertheless important for the final diagnosis and accurate management of the diseases. Targeted next-generation sequencing is a rapid and cost-effective method to sequence large numbers of genes simultaneously. We developed a targeted next-generation sequencing assay, MyoCap, for the coding exons and UTRs of 180 myopathy related genes including 42 novel genes that have not yet been associated with myopathies. DNA samples of four controls with known mutations and 61 patients negative for previous candidate gene approaches were sequenced. The genetic defect was totally or partly clarified in 21 patients with nine of them having potential disease-causing mutations in TTN. MyoCap provides higher read depth and coverage with lower price in the myopathy related genes compared to the whole exome sequencing and is thus very suitable for diagnostic use.