Next-generation Sequencing Platforms Research Articles

P-540 Strategic implementation of PGT-M for multiple genes post qhole exome sequencing in couples with familial history: a comprehensive study

Abstract Study question What advantages does Whole Exome Sequencing offer to couples with a familial history of hereditary disorders? Summary answer WES offers a comprehensive risk assessment, facilitating the development of an effective PGT-M for Multiple Genes in the prevention of severe diseases in their offspring What is known already PGT-M enables the transfer of unaffected embryos to couples at risk of transmitting monogenic disorders. With the advancements in technologies like Whole Exome Sequencing (WES) and Whole Genome Sequencing (WGS) identifying causal mutations, there is a growing demand for PGT-M applications that concurrently assess multiple genetic disorders. This is particularly crucial, as families initially referred for one condition may later be discovered to be at risk for additional monogenic disorders due to the common carriership status of the couples. Study design, size, duration This is a retrospective study including couples with previous familial history of genetic disorders referred to a single genetic unit between years 2020-2024. Either WES-DUO or WES-TRIO analysis were performed in the absence or presence of an affected child, respectively. Participants/materials, setting, methods WES-DUO or WES-Trio analyses were performed on 94 couples with a familial history of genetic diseases encompassing diverse medical domains. Around 50% of the couples exhibited consanguinity. Utilizing the Novaseq 6000 next-generation sequencing platform, exonic regions were sequenced with an average coverage of 100X. Bioinformatic analysis followed the manufacturer’s recommended workflow, and variant analysis adhered to ACMG criteria, ClinVar, and HGMD. Segregation analysis was conducted through Sanger sequencing using region-specific primers. Main results and the role of chance WES, followed by segregation analysis, pinpointed reportable pathogenic or likely pathogenic variants in 46 out of 94 families as the root cause of the referral condition. Interestingly, 42 of these families exhibited at least one additional common gene mutation in both partners. Notably, among the 27 families with confirmed diagnoses, in vitro fertilization (IVF) and preimplantation genetic testing for monogenic disorders (PGT-M) were undertaken. Within this cohort, PGT-M for multiple genes was successfully conducted in 22 families, 96 embryos were found to be transferable resulting in the birth of healthy infants. Limitations, reasons for caution The overall diagnostic confirmation rate of WES analysis reached 48.9%. A quite high diagnostic yield achieved in this study is notably influenced by the substantial prevalence of consanguineous cases. Wider implications of the findings This study highlights the need for implementing PGT-M for multiple genes. Failure to undergo such testing would potentially lead to the birth of children affected by other conditions.This study also underscores the efficiency of encompassing WES, mutation confirmation, segregation analysis, bioinformatic processing and PGT applications in a single genetic unit. Trial registration number none

Laboratory validation and clinical utility of next-generation sequencing-based IGH/TCR clonality testing for the monitoring of measurable residual disease in acute lymphoblastic leukaemia: real-world experience at Austin Pathology

Measurable residual disease (MRD) testing is an essential aspect of disease prognostication in acute lymphoblastic leukaemia (ALL) and informs clinical decisions. The depth of MRD clearance is highly relevant and requires assays with sufficient sensitivity. Austin Pathology is one of the few laboratories in Australia currently utilising a fully validated and National Association of Testing Authorities (NATA)-accredited ultrasensitive next-generation sequencing (NGS) platform for MRD monitoring in ALL. This technology is based on the detection of clonal rearrangement of immunoglobulin and T cell receptor genes in leukaemic cells, and is capable of achieving a limit of detection at least one to two logs below that of multiparametric flow cytometry (MFC). In this retrospective analysis, we report a clonotype detection rate of up to 85.7% at diagnosis, and a concordance rate of 78.7% in MRD results between NGS and MFC. Of the discordant samples, nearly all were NGS+/MFC-, highlighting the superior sensitivity of NGS. The enhanced sensitivity is clinically relevant, as discordant MRD results often heralded fulminant relapse, and therefore offer clinicians additional lead time and a window of opportunity to initiate pre-emptive therapy. Notwithstanding a small and heterogeneous cohort, our real-world survival data indicate an intermediate relapse risk for NGS+/MFC- patients. In light of recent approval of Medicare rebatable ALL MRD testing, we discuss how NGS can complement other techniques such as MFC in personalising management strategies. We recommend routine clonality testing by NGS at diagnosis and use a multi-modality approach for subsequent MRD monitoring.

Advancing Clinical Microbiology: Applications and Future of Next-Generation Sequencing

Next-generation sequencing (NGS) has reformed infectious disease management, including COVID-19. While real-time polymerase chain reaction (PCR) is widely used for rapid pathogen detection, it requires predefined targets. NGS offers an unbiased approach, detecting multiple pathogens simultaneously without prior knowledge. Despite its potential, NGS implementation in clinical settings faces challenges like high costs and technical complexity. NGS platforms like Illumina, Ion Torrent, and Nanopore provide high-throughput sequencing, identifying pathogens and resistance markers. Applications include whole genome sequencing (WGS), metagenomic NGS (mNGS), and targeted NGS (tNGS). Integrating NGS with conventional methods could improve diagnostics, but current evidence is mixed for supporting its widespread clinical use.

AMLdb: a comprehensive multi-omics platform to identify biomarkers and drug targets for acute myeloid leukemia.

Acute myeloid leukemia (AML) is one of the leading leukemic malignancies in adults. The heterogeneity of the disease makes the diagnosis and treatment extremely difficult. With the advent of next-generation sequencing (NGS) technologies, exploration at the molecular level for the identification of biomarkers and drug targets has been the focus for the researchers to come up with novel therapies for better prognosis and survival outcomes of AML patients. However, the huge amount of data from NGS platforms requires a comprehensive AML platform to streamline literature mining efforts and save time. To facilitate this, we developed AMLdb, an interactive multi-omics platform that allows users to query, visualize, retrieve, and analyse AML related multi-omics data. AMLdb contains 86 datasets for gene expression profiles, 15 datasets for methylation profiles, CRISPR-Cas9 knockout screens of 26 AML cell lines, sensitivity of 26 AML cell lines to 288 drugs, mutations in 41 unique genes in 23 AML cell lines, and information on 41 experimentally validated biomarkers. In this study, we have reported five genes, i.e. CBFB, ENO1, IMPDH2, SEPHS2, and MYH9 identified via our analysis using AMLdb. ENO1 is uniquely identified gene which requires further investigation as a novel potential target while other reported genes have been previously confirmed as targets through experimental studies. Top of form we believe that these findings utilizing AMLdb can make it an invaluable resource to accelerate the development of effective therapies for AML and assisting the research community in advancing their understanding of AML pathogenesis. AMLdb is freely available at https://project.iith.ac.in/cgntlab/amldb.

Gene fusions: A retrospective analysis of therapies and treatments of MTB patients over 7 years.

e15183 Background: Gene fusions (GF) are less common than single nucleotide variants, amplifications, and deletions, but have been suggested to be more predictive of responses to specific molecularly targeted therapies than other types of molecular alterations. We reviewed our institutional trends for overall survival in our molecular tumor board database based on the receipt of molecularly targeted therapy. Methods: We identified patients with non-hematologic malignancies and gene fusions (GF) identified by DNA based next generation sequencing (NGS) and reviewed by the Inova Schar Cancer Institute Molecular Tumor Board (ISCI MTB) between March 1, 2016 and March 1, 2023. GFs were confirmed by re-evaluation of next generation sequencing, and we determined the number of patients who received molecularly targeted therapy (RTT) specific for the GF. We evaluated overall survival (OS) in the RTT cohort and in the cohort that did not receive targeted therapy (NTT). TMPRSS fusions in prostate cancer were excluded from this analysis. Results: There were 93 patients with gene fusions in advanced staged cancers that met study criteria. Twenty-three were lost to follow up, transferred care outside ISCI, or enrolled in hospice. Of 70 stage IV patients reviewed, thirty-seven were found to have received GF targeted therapy. OS for all patients was 28.5 months. Patients in the RTT cohort had a median OS of 45 months and patients in the NTT cohort had an OS of 26.8 months (p-value = 0.16, Log-rank test). Conclusions: Targeted therapy in solid tumors with gene fusions is associated with prolonged overall survival. It is critical for MTBs to use NGS platforms that identify GF. Further studies examining OS and GF are warranted. [Table: see text]

Genomic evolution of patients with myeloid neoplasms and known antecedent clonal hematopoiesis.

6584 Background: Clonal hematopoiesis (CH) refers to the clonal expansion of hematopoietic stem cells (HSCs) with one or more somatic mutations in the absence of a hematologic malignancy. Although it is associated with normal aging, CH confers an increased risk of several comorbidities and hematologic malignancies, including myeloid neoplasms (MNs). However, the outcomes of patients with CH that transformed to MN are unknown. Here, we aim to evaluate the clinicopathologic characteristics and survival outcomes of patients with MN with known antecedent CH. Methods: We retrospectively evaluated patients seen at a tertiary cancer center from May 2016 to July 2023 with myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), or acute myeloid leukemia (AML) and identified those who had CH prior to MN diagnosis. At the time of MN transformation, genomic data was extracted from whole bone marrow aspirate samples and subject to 81-gene target PCR-based sequencing using a next-generation sequencing platform. Results: A total of 36 patients were identified; at the time of CH diagnosis, 79% had known cardiovascular comorbidities and 81% had a prior diagnosis of non-myeloid malignancies for which 72% received prior chemotherapy and 25% radiation. According to the Clonal Hematopoiesis Risk Score (CHRS), 22% were low, 56% deemed intermediate, and 22% at high risk of transformation to MNs. The most frequently observed CH-associated mutations were TET2 (41%), TP53 (38%), IDH2 (16%), EZH2 (16%), ASXL1 (13%), and DNMT3A (13%); 69% had more than one somatic mutation, and 61% had normal cytogenetics. The median time to transformation to MN was 15 months, 26 months, and 24 months for MDS (n=28), CMML (n=2), and AML (n=6) patients, respectively (p=0.72). The median overall survival from MN diagnosis was 54 months, not reached, and 2 months for MDS, CMML, and AML, respectively (p=0.0015). We further delved into the genomic evolution from CH to MN. In the 6 pts who transformed to AML, 4 had a TP53 mutation at CH with expansion of the clone at MN transformation, 2 acquired NPM1 mutations and 1 acquired FLT3 mutations. Both pts who progressed to CMML had expansion of their underlying CH clones (KRAS and ZRSR2). In those who developed MDS, 22 pts had expansion of their CH-mutated clone at the time of MN, most frequently in TET2 (36%), IDH2 (18%), and TP53 (14%); 5 pts acquired new driver mutations outside of their CH clone; and 1 pt lost their CH clone with no new somatic mutations detected. Conclusions: Individuals with CH are at risk of progressing to MN, especially with external selective pressures like antineoplastic therapy that are not accounted for in prognostic systems like CHRS. Patients often have underlying high-risk clones at the time of CH diagnosis that expand at the time of MN, though some patients acquired new driver mutations.

Genetic landscape for majority and minority HIV-1 drug resistance mutations in antiretroviral therapy naive patients in Accra, Ghana

BackgroundThe successful detection of drug-resistance mutations (DRMs) in HIV-1 infected patients has improved the management of HIV infection. Next-generation sequencing (NGS) to detect low-frequency mutations is predicted to be useful for efficiently testing minority drug resistance mutations, which could contribute to virological failure. This study employed Sanger sequencing and NGS to detect and compare minority and majority drug resistance mutations in HIV-1 strains in treatment-naive patients from Ghana. MethodFrom a previous study, 20 antiretroviral therapy (ART)-naive participants were selected for a cross-sectional study. Sanger sequencing and NGS techniques were used to detect the majority and minority HIV drug resistance (HIVDR) mutations, respectively, in the protease (PR) and partial reverse transcriptase (RT) genes. NGS detected mutations at 1 % and 5 % frequencies and Sanger sequencing at ≥20 % frequencies. The sequences obtained from NGS and Sanger sequencing platforms were submitted to the Stanford HIV drug resistance database for subtyping, mutation identification, and interpretations. ResultsSequences from the twenty participants where the CRF02_AG was the predominant strain (16, 80 %) were analyzed. NGS detected 25 mutations in the RT and PR genes, compared to 21 mutations by Sanger sequencing. Minority DRMs were detected at the prevalence of 55.0 % with NGS against 35 % DRMs by Sanger sequencing. One of the patients had eight different HIVDR variants, with two minority variants. These mutations were directed against PI (K20I and D30DN), NNRTI (Y181C, M23LM and V108I) and NRTI (K65R, M184I, and D67N). ConclusionThe study affirms the usefulness of genomic sequencing for drug resistance testing in HIV. It further shows that Sanger sequencing alone may not be adequate to detect mutations and that NGS capacity should be developed and deployed in the Ghanaian clinical settings for patients living with HIV.

Predicting pathological response to neoadjuvant immunotherapy plus chemotherapy in resectable non-small cell lung cancer through liquid biopsy.

e20073 Background: Neoadjuvant immunotherapy (ICI) plus chemotherapy (CT) provides a significant benefit compared to CT alone in resectable non-small cell lung cancer (NSCLC). However, further research is needed to identify the populations benefitting from this combined strategy. Methods: Patients (pts) diagnosed with stage II-III resectable NSCLC who opted for neoadjuvant ICI plus CT were enrolled. Plasma samples were collected before treatment initiation (T1) and before surgery (T2). A next-generation sequencing (NGS) platform spanning 769 cancer-related genes was utilized to detect circulating tumor DNA (ctDNA). The ctDNA concentration was presented as haploid genome equivalents (hGE) per mL of plasma. Additionally, low-pass whole-methylome sequencing was employed to explore three features of cell-free DNA, including methylated fragment ratio (MFR), fragment size index (FSI), and chromosomal aneuploidy of featured fragments (CAFF). Clinical and pathological data was obtained from medical records. Results: Out of 24 pts enrolled, 18 pts were evaluable on both platforms. The major pathological response (MPR) rate was 70.8% (17/24), and the pathological complete response (pCR) rate was 37.5% (9/24). Squamous cell carcinoma (SCC) showed a higher MPR rate than adenocarcinoma (P = 0.014). Pts with clinical T stage 2 showed a trend toward a higher MPR rate than those with T stage 3/4 (P = 0.069). No single molecular biomarker was found to be independently associated with MPR or pCR at T1. A linear discriminant model was constructed based on clinical T stage, MFR, and hGE at T1, which predicted both MPR and pCR with an accuracy of 83.3% (n = 18). In pts with SCC (n = 15), the accuracy of this model in predicting MPR and pCR was 80.0% and 86.7%, respectively. Moreover, the CAFF score at T2 was significantly higher in non-MPR pts than in MPR pts (P = 0.003). The dynamic change of CAFF score [calculation: (T2-T1) / T1] was also significantly related to MPR (P = 0.006). Conclusions: Baseline cfDNA features and clinical parameters effectively predicted pathological response of neoadjuvant ICI plus CT in resectable NSCLC, which could potentially serve as a tool for identifying patients that would benefit most from the combined approach.

Utility of an amplicon-based ctDNA platform for advanced cholangiocarcinomas (CCA) in Asia.

e16206 Background: Cholangiocarcinoma represents a major cause of cancer mortality in Asia. While clinical outcomes have improved with the addition of anti-PD1/PDL1 agents to standard-of-care chemotherapy for advanced disease, recent data suggests that novel agents against IDH1 mutations, FGFR2 and NTRK rearrangements, BRAF V600E mutations, and microsatellite instability (MSI) are effective. Tumor-based next generation sequencing (NGS) is routinely recommended by guidelines but is limited by tissue availability and quality in patients with unresectable disease. A comprehensive circulating tumor DNA-based (ctDNA) NGS platform offers an acceptable alternative, but its clinical utility and pickup rate for oncogenic drivers remain unknown. Methods: In our multi-national cohort of advanced CCA across 5 countries, we explored the utility of Lucence LiquidHALLMARK (LHM), a Medicare-reimbursed, amplicon-based NGS ctDNA platform targeting 80 cancer related genes, 10 fusions, and MSI. Performance of LHM has been previously reported. Data was retrieved in-house by a de-identified centralized database and pooled for analysis. Here, we report the demographic and genomic findings of our cohort and highlight the impact of testing on actionable molecular alteration discovery. Results: Between Jan 2020 and Dec 2023, 60 samples from 56 patients (62.5% male) with advanced CCA in Hong Kong SAR (50.0%), Singapore (41.1%), Thailand (3.6%), Philippines (3.6%), and Malaysia (1.8%) underwent LHM testing. Metastatic disease accounted for 83.9% and 8.9% had locally advanced disease. Median age was 67 years (range 33-88), with 34% and 30% of tests obtained during first-line therapy and at diagnosis respectively. Only 6 cases had available tissue NGS results. Analysis on 56 baseline samples showed an 80.4% discovery rate (45/56 patients) for ≥1 molecular alterations. A median of 2 variants (mean 3.27) was detected per sample, with variant allele fraction ranging from 0.05% to 66.5%. Importantly, 8.9% (5/56) of patients harbored ≥1 actionable variants, including 3 with FGFR2 rearrangements ( FGFR2- WAC, FGFR2- ACTR1A, FGFR2- CAMK2D) and 2 with BRAF V600E drivers. A further 3 patients had potentially druggable variants in IDH2 and FGFR3 and 12 harbored BRCA1/2 variants, of which 4 were deleterious. None of the samples had MSI. Top 10 mutated genes were TP53 (48%), KRAS (21%), BRCA2 (18%), PIK3CA (11%), TERT (9%), CDKN2A (9%), SMAD4 (9%), FGFR2 (7%), BRAF (7%), and CDK6 (5%). Limited histopathological and treatment outcomes are available in the current dataset. Conclusions: Our advanced cholangiocarcinoma cohort of 56 patients from 5 Asian countries showed that an amplicon based ctDNA liquid biopsy platform overcomes the tissue-related challenges of genomic profiling specific to unresectable biliary tumors. We showed an 80.4% discovery rate for any molecular alterations and a 8.9% pickup rate for variants with strong actionability.

The performance of homopolymer detection using dichromatic and tetrachromatic fluorogenic next-generation sequencing platforms

ObjectivesHomopolymer (HP) sequencing is error-prone in next-generation sequencing (NGS) assays, and may induce false insertion/deletions and substitutions. This study aimed to evaluate the performance of dichromatic and tetrachromatic fluorogenic NGS platforms when sequencing homopolymeric regions.ResultsA HP-containing plasmid was constructed and diluted to serial frequencies (3%, 10%, 30%, 60%) to determine the performance of an MGISEQ-2000, MGISEQ-200, and NextSeq 2000 in HP sequencing. An evident negative correlation was observed between the detected frequencies of four nucleotide HPs and the HP length. Significantly decreased rates (P < 0.01) were found in all 8-mer HPs in all three NGS systems at all four expected frequencies, except in the NextSeq 2000 at 3%. With the application of a unique molecular identifier (UMI) pipeline, there were no differences between the detected frequencies of any HPs and the expected frequencies, except for poly-G 8-mers using the MGI 200 platform. UMIs improved the performance of all three NGS platforms in HP sequencing.ConclusionsWe first constructed an HP-containing plasmid based on an EGFR gene backbone to evaluate the performance of NGS platforms when sequencing homopolymeric regions. A highly comparable performance was observed between the MGISEQ-2000 and NextSeq 2000, and introducing UMIs is a promising approach to improve the performance of NGS platforms in sequencing homopolymeric regions.

Whole-genome sequencing of marine water-derived Curvularia verruculosa KHW-7: a pioneering study

Marine microorganisms are renowned for being a rich source of new secondary metabolites that are significant to humans. The fungi strain KHW-7 was isolated from the seawater collected from the Gulf of Khambhat, India, and identified as Curvularia verruculosa KHW-7. On a next-generation sequencing platform, C. verruculosa KHW-7’s whole-genome sequencing (WGS) and gene annotation were carried out using several bioinformatic methods. The 31.59 MB genome size, 52.3% GC, and 158 bp mean read length were discovered using WGS. This genome also contained 9,745 protein-coding genes, including 852 secreted proteins and 2048 transmembrane proteins. The antiSMASH algorithm used to analyze genomes found 25 secondary metabolite biosynthetic gene clusters (BGCs) that are abundant in terpene, non-ribosomal peptide synthetase (NRPS), and polyketides type 1 (T1PKS). To our knowledge, this is the first whole-genome sequence report of C. verruculosa. The WGS analysis of C. verruculosa KHW-7 indicated that this marine-derived fungus could be an efficient generator of bioactive secondary metabolites and an important industrial enzyme, both of which demand further investigation and development.

Detection of tumor-derived cell-free DNA in cerebrospinal fluid using a clinically validated targeted sequencing panel for pediatric brain tumors.

Clinical sequencing of tumor DNA is necessary to render an integrated diagnosis and select therapy for children with primary central nervous system (CNS) tumors, but neurosurgical biopsy is not without risk. In this study, we describe cell-free DNA (cfDNA) in blood and cerebrospinal fluid (CSF) as sources for "liquid biopsy" in pediatric brain tumors. CSF samples were collected by lumbar puncture, ventriculostomy, or surgery from pediatric patients with CNS tumors. Following extraction, CSF-derived cfDNA was sequenced using UW-OncoPlex™, a clinically validated next-generation sequencing platform. CSF-derived cfDNA results and paired plasma and tumor samples concordance was also evaluated. Seventeen CSF samples were obtained from 15 pediatric patients with primary CNS tumors. Tumor types included medulloblastoma (n = 7), atypical teratoid/rhabdoid tumor (n = 2), diffuse midline glioma with H3 K27 alteration (n = 4), pilocytic astrocytoma (n = 1), and pleomorphic xanthoastrocytoma (n = 1). CSF-derived cfDNA was detected in 9/17 (53%) of samples, and sufficient for sequencing in 8/10 (80%) of extracted samples. All somatic mutations and copy-number variants were also detected in matched tumor tissue, and tumor-derived cfDNA was absent in plasma samples and controls. Tumor-derived cfDNA alterations were detected in the absence of cytological evidence of malignant cells in as little as 200µl of CSF. Several clinically relevant alterations, including a KIAA1549::BRAF fusion were detected. Clinically relevant genomic alterations are detectable using CSF-derived cfDNA across a range of pediatric brain tumors. Next-generation sequencing platforms are capable of producing a high yield of DNA alterations with 100% concordance rate with tissue analysis.

Simultaneous sequencing of 102 Y-STRs on Ion Torrent ™ GeneStudio ™ S5 System

The Precision ID NGS System from Thermo Fisher Scientific is a mainstream next-generation sequencing (NGS) platform used in forensic laboratories to detect almost all commonly used forensic markers, except for Y-chromosomal short tandem repeats (Y-STRs). This study aimed to: 1) develop a Y-STR panel compatible with the automatic workflow of the NGS system using Ion AmpliSeq Technology, 2) evaluate the panel performance following the SWGDAM guidelines, and 3) explore the possibility of using a combination workflow to detect autosomal STRs and Y-STRs (AY-STR NGS workflow). The GrandFiler Y-STR Panel was successfully designed using the ‘separating’ and ‘merging’ strategies, including 102 Y-STRs and Amelogenin with an average amplicon length of 133 bp. It is a mega Y-STR multiplex system in which up to 16 samples can be sequenced simultaneously on an Ion 530 ™ Chip. Developmental validation studies of the performance of the NGS platform, species specificity, reproducibility, concordance, sensitivity, degraded samples, case-type samples, and mixtures were conducted to unequivocally determine whether the GrandFiler Y-STR Panel is suitable for real scenarios. The newly developed Y-STR panel showed compelling run metrics and NGS performance, including 92.47% bases with ≥ Q20, 91.80% effective reads, 2106 × depth of coverage (DoC), and 97.09% inter-locus balance. Additionally, it showed high specificity for human males and 99.40% methodological and bioinformatical concordance, generated complete profiles at ≥ 0.1 ng input DNA, and recovered more genetic information from severely degraded and diverse case samples. Although the outcome when used on mixtures was not as expected, more genetic information was obtained compared to that from capillary electrophoresis (CE) methods. The AY-STR NGS workflow was established by combining the GrandFiler Y-STR Panel with the Precision ID GlobalFiler ™ NGS STR Panel v2 at a 2:1 concentration ratio. The combination workflow on NGS performance, reproducibility, concordance, and sensitivity was as stable as the single Y-STR NGS workflow, providing more options for forensic scientists when dealing with different case scenarios. Overall, the GrandFiler Y-STR Panel was confirmed as the first to effectively detect a large number of Y-STR markers on the Precision ID NGS System, which is compatible with 51 Y-STRs in commercial CE kits and 51 Y-STRs in commercial NGS kits and the STRBase. The panel is as robust, reliable, and sensitive as current CE/NGS kits, and is suitable for solving real cases, especially for severely degraded samples (degradation index > 10).

Genetic diversity and population structure of four Anatolian sheep revealed by genome-wide ddRADseq data

This is the first wide-scale study to reveal next-generation sequencing-based and comprehensive study to reveal genetic diversity, population structure, and phylogenetic relationships of some of Türkiye's indigenous fat-tailed sheep breeds. A total of 80 animals belonging to Akkaraman (AKR), Güney Karaman (GKR), Morkaraman (MKR), and Karakaş (KRK) sheep breeds were genotyped with genome-wide of 296.097 single nucleotide polymorphisms (SNPs) data recovered from double digest restriction site-associated DNA sequencing (ddRADseq) libraries processed by Illumina HiSeq X Ten instrument. Minor allele frequency (MAF) ranged from 0.311 (MKR) to 0.316 (AKR and GKR). Observed heterozygosity (HO) values were higher than expected values (HE) across all populations with a mean of 0.307. Negative inbreeding coefficient (FIS) values were observed in four sheep populations varying between −0.034 (AKR) to −0.060 (MKR). Cross-entropy criterion-based ADMIXTURE analysis demonstrated that four Anatolian sheep breeds were represented by two ancestral populations in which GKR and AKR were distinct, while KRK and MKR were mixed populations. Genetic distance-based tree, discriminant analysis of principal components (DAPC), and ADMIXTURE analyses confirmed that GKR, which is believed to be derived from the AKR breed, has become genetically different from other sheep populations. Moreover, no migration from and into the GKR breed was detected, while a migration edge was drawn from the KRK and MKR clade into the AKR breed by the TreeMix algorithm. This study confirmed that high-density SNP data obtained from advanced next-generation sequencing (NGS) platforms are efficient in genetically distinguishing animal breeds, even varieties. It is recommended that further NGS-based studies could be carried out to better characterize different indigenous livestock populations raised in Türkiye.

Microfluidics-based EGFR mutation detection and its implication in the resource-limited clinical setting.

Management of lung cancer today obligates a mutational analysis of the epidermal growth factor receptor (EGFR) gene particularly when Tyrosine Kinase Inhibitor (TKI) therapy is being considered as part of prognostic stratification. This study evaluates the performance of automated microfluidics-based EGFR mutation detection and its significance in clinical diagnostic settings. Formalin-fixed, paraffin-embedded (FFPE) samples from NSCLC patients (n = 174) were included in a two-phase study. Phase I: Validation of the platform by comparing the results with conventional real-time PCR and next-generation sequencing (NGS) platform. Phase II: EGFR mutation detection on microfluidics-based platform as part of routine diagnostics workup. The microfluidics-based platform demonstrates 96.5% and 89.2% concordance with conventional real-time PCR and NGS, respectively. The system efficiently detects mutations across the EGFR gene with 88.23% sensitivity and 100% specificity. Out of 144 samples analysed in phase II, the platform generated valid results in 94% with mutation detected in 41% of samples. This microfluidics-based platform can detect as low as 5% mutant allele fractions from the FFPE samples. Therefore the microfluidics-based platform is a rapid, complete walkaway, with minimum tissue requirement (two sections of 5 μ thickness) and technical skill requirement. The method can detect clinically actionable EGFR mutations efficiently and can be considered a reliable diagnostic platform in resource-limited settings. From receiving samples to reporting the results this platform provides accurate data without much manual intervention. The study helped to devise an algorithm that emphasizes effective screening of the NSCLC cases for EGFR mutations with varying tumour content. Thus it helps in triaging the cases judiciously before proceeding with multigene testing.

Circulating tumor DNA- and cancer tissue-based next-generation sequencing reveals comparable consistency in targeted gene mutations for advanced or metastatic non-small cell lung cancer.

Molecular subtyping is an essential complementarity after pathological analyses for targeted therapy. This study aimed to investigate the consistency of next-generation sequencing (NGS) results between circulating tumor DNA (ctDNA)-based and tissue-based in non-small cell lung cancer (NSCLC) and identify the patient characteristics that favor ctDNA testing. Patients who diagnosed with NSCLC and received both ctDNA- and cancer tissue-based NGS before surgery or systemic treatment in Lung Cancer Center, Sichuan University West China Hospital between December 2017 and August 2022 were enrolled. A 425-cancer panel with a HiSeq 4000 NGS platform was used for NGS. The unweighted Cohen's kappa coefficient was employed to discriminate the high-concordance group from the low-concordance group with a cutoff value of 0.6. Six machine learning models were used to identify patient characteristics that relate to high concordance between ctDNA-based and tissue-based NGS. A total of 85 patients were enrolled, of which 22.4% (19/85) had stage III disease and 56.5% had stage IV disease. Forty-four patients (51.8%) showed consistent gene mutation types between ctDNA-based and tissue-based NGS, while one patient (1.2%) tested negative in both approaches. Advanced diseases and metastases to other organs would be fit for the ctDNA-based NGS, and the generalized linear model showed that T stage, M stage, and tumor mutation burden were the critical discriminators to predict the consistency of results between ctDNA-based and tissue-based NGS. ctDNA-based NGS showed comparable detection performance in the targeted gene mutations compared with tissue-based NGS, and it could be considered in advanced or metastatic NSCLC.

Rapid Detection and Quick Characterization of African Swine Fever Virus Using the VolTRAX Automated Library Preparation Platform.

African swine fever virus (ASFV) is the causative agent of a severe and highly contagious viral disease affecting domestic and wild swine. The current ASFV pandemic strain has a high mortality rate, severely impacting pig production and, for countries suffering outbreaks, preventing the export of their pig products for international trade. Early detection and diagnosis of ASFV is necessary to control new outbreaks before the disease spreads rapidly. One of the rate-limiting steps to identify ASFV by next-generation sequencing platforms is library preparation. Here, we investigated the capability of the Oxford Nanopore Technologies' VolTRAX platform for automated DNA library preparation with downstream sequencing on Nanopore sequencing platforms as a proof-of-concept study to rapidly identify the strain of ASFV. Within minutes, DNA libraries prepared using VolTRAX generated near-full genome sequences of ASFV. Thus, our data highlight the use of the VolTRAX as a platform for automated library preparation, coupled with sequencing on the MinION Mk1C for field sequencing or GridION within a laboratory setting. These results suggest a proof-of-concept study that VolTRAX is an effective tool for library preparation that can be used for the rapid and real-time detection of ASFV.

Fungi associated with fine roots of Fraxinus excelsior affected by ash dieback detected by next-generation sequencing

AbstractThe decline of European ash by dieback caused by Hymenoscyphus fraxineus together with stem collar necroses and rots caused by various fungi has been investigated intensively during the last years. Nevertheless, hitherto nearly nothing is known about the species diversity of the fungal rhizobiome of ash trees. Here we investigated the fine roots of affected ash trees on 15 sampling sites in 6 federal countries of Germany. Fine-root samples have been treated in three different sample regimes each as root-adhering soil, unsterilized fine roots and sterilized fine roots. The samples of trees in sampling sites were pooled to get an overview of the species-richness in the area. The next-generation sequencing platform Oxford Nanopore MinION was used to sequence the entire ITS of pooled probes. Most abundant phyla in all samples were the Basidiomycota and Ascomycota. Species richness in sterilized roots was significantly different from unsterilized roots and root-adhering soil. Surprisingly most abundant genera in sterilized roots were the genera Mycena, Mycenella and Delicatula, all of them agaricoids with saprophytic lifestyle. Eleven genera of Glomeromycota have been detected in various abundances, whereas the detection of H. fraxineus was neglectable.

Abstract PS11-03: Comparison of next-generation sequencing (NGS) results from the cerebrospinal fluid, peripheral blood, and systemic metastatic tumor tissue of patients with metastatic breast cancer (MBC) and leptomeningeal disease (LMD)

Abstract Introduction. Leptomeningeal disease (LMD) is a devastating complication of advanced malignancies and occurs in 5-15% of patients (pts) with metastatic breast cancer (MBC). Molecular characterization of the primary tumor to identify actionable mutations is standard of care, and targeted therapies have significantly improved survival. In contrast, metastatic tumors to the central nervous system (CNS) are not routinely assessed for the presence of actionable mutations, so the frequency and concordance of actionable mutations centrally vs. peripherally is not well characterized. In this single-center prospective study, we collected cerebrospinal fluid (CSF) in pts with known or suspected LMD and performed next generation sequencing (NGS) to evaluate the status of actionable mutations in the CSF and compared these results with NGS from matched peripheral blood and systemic metastatic tumor samples. Methods. We enrolled sixteen (16) pts with MBC and suspected or confirmed LMD at a single academic center from 2021-2023 in this non-therapeutic prospective study and collected CSF, peripheral blood, and archival systemic tumor samples from each pt. We used NGS to evaluate the status of actionable biomarkers in the CSF, blood, and tissue using the following NGS platforms: CNSide (Biocept) for CSF; Guardant 360, Foundation One CDx and CNSide for blood; and Caris and the UCSF CLIA-validated UCSF500 panel for the systemic metastatic tumor samples. We also evaluated CSF tumor cell enumeration and HER2 status in a subset of pts (CNSide). Results. Of the sixteen pts with MBC and suspected or confirmed LMD, 81% (13/16) were ultimately determined to be LMD-positive based on positive CSF cytology and/or convincing MRI imaging, including 6 pts with HR+/HER2- MBC, 6 pts with metastatic triple negative breast cancer (mTNBC), and 1 pt with HR+/HER2+ MBC; 3 pts were determined to be LMD-negative based on similar criteria. Of the 6 pts with HR+/HER2- MBC and LMD, NGS detected actionable mutations in the CSF in 67% (4/6), including two pts with PIK3CA E545E, one pt with both PIK3CA E542K and ERBB2 amplification, and 1 pt with ESR1 Y537S. Matched NGS analysis of the peripheral blood and/or systemic metastatic tissue showed concordance with PIK3CA E542K detected across tissue types in two pts, but discordance across tissue types in the other pts, including ESR1 detected systemically in two pts but not in the CSF. Of the 6 pts with mTNBC and LMD, CSF NGS detected actionable mutations in the CSF in 17% (1/6) of pts, including one pt with PIK3CA E542K; analysis of blood NGS for this pt is pending. 3 pts (50%) had mutations in TP53 in the CSF (1 pt with R273H and two pts with K132R), which were concordant across tissue types in all 3 pts. NGS did not detect any variants in the CSF of the one pt with HR+/HER2+ MBC and LMD. CSF tumor cell enumeration and central HER2 status will also be reported. Conclusion. This study demonstrates that NGS can detect actionable mutations in the CSF of pts with MBC and LMD. We observed concordance and heterogeneity in the status of actionable mutations between the CSF, peripheral blood, and systemic metastatic tumor tissue. Larger studies are needed to assess the clinical utility of these observations, particularly with the development of several novel targeted agents that are CNS-penetrant. Citation Format: Laura Huppert, Lindy Her, Christine Hodgdon, Susie Brain, Carol Simmons, Jo Chien, Melanie Majure, Hope Rugo, Mark Jesus Magbanua, Ron Balassanian, Michelle Melisko. Comparison of next-generation sequencing (NGS) results from the cerebrospinal fluid, peripheral blood, and systemic metastatic tumor tissue of patients with metastatic breast cancer (MBC) and leptomeningeal disease (LMD) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS11-03.

Abstract PO2-04-06: Investigation of the genomic evolution of HER2-positive breast cancer following progression on dual HER2-targetted therapy with Trastuzumab and Tucatinib

Abstract Background: Advanced HER2-positive breast cancer remains an incurable and highly morbid condition. One of the key challenges has been treatment of central nervous system (CNS) metastases, commonly seen in those with HER2-positive disease. Tucatinib is a recently approved treatment HER2-targetting tyrosine kinase inhibitor with proven CNS activity. There has been little analysis of potential genomic mechanisms of drug resistance. Method: We retrospectively identified patients that were treated with tucatinib, capecitabine and trastuzumab. We collated baseline clinical data and treatment history and all tumour and plasma samples available were obtained for testing. DNA was extracted from plasma samples using the QIAamp DNA Mini Kit (Qiagen) on 4ml plasma samples. Targeted next generation sequencing (NGS) of serial tumour and plasma samples was performed using a custom hybrid capture assay that covered 180 genes known to be recurrently mutated in breast cancer focusing on the known genomic landscape of metastatic disease as well as the inclusion of specific actionable targets. Survival analyses was performed using Cox regression models. Results: We identified 11 patients at our institution. Plasma samples were available for all 11 patients with a total of 107 serial samples across multiple time points, with a median of 8 (range 2-19) samples per patient. All 11 patients had baseline and End of Treatment (EOT) samples. In addition, 6/11 (54%) patients had tumour sequenced on the same NGS platform prior to commencement of therapy, with 12 individual tumour samples tested. The median PFS of our cohort was 9.6 months (4.0 - NR) with a median OS of 36.2 months (10.4-NR). 7/11 (64%) patients had CNS disease at baseline with their median PFS being 13.5 months (3.9-24.4) and median OS of 36.2months (10.4-NR). Overall, we observed an average of 18.4 (4-35) mutations per patient in tumour, 5.1 (1-15) in baseline plasma and 5.8 (2-14) in EOT plasma. The most frequent mutations seen in baseline tumour samples were ERBB2 (92%), KMT2C (67%) and NCOR1 (58%) compared with KMT2C (64%), NOTCH4 (36%) and PIK3CA (27%) in baseline plasma. Patients with a lower number of ctDNA mutations at baseline showed a trend towards improved survival (PFS 14 months vs 7.5 months). In this small cohort of patients we identified a higher than expected rates of KMT2C mutations. A KMT2C mutation was identified in 67% of samples which is higher than previously described rates of 7-15% in TCGA and METABRIC cohorts respectively. The presence of a concordant ctDNA KMT2C mutation in baseline and progression samples was a poor prognostic indicator (mPFS 7.5 months) when compared with no KMT2C mutation (mPFS 15.5 months) p =0.14 suggesting a possible mechanism of resistance to tucatinib. Conclusions: In this retrospective analysis of patients treated with Tucatinib, Trastuzumab and Capecitabine we established the genomic landscape of heavily pre-treated, advanced HER2-positive disease is complex. We observed there may be a correlation with KMT2C mutations and poorer clinical outcomes, however given the very small number of patients this data remains hypothesis generating, requiring validation with larger datasets. Citation Format: Elizabeth Blackley, Courtney van Geelen, Yi-An Ko, Stephen Wong, Miriam Yeung, Stephen Luen, Sarah-Jane Dawson, Sherene Loi. Investigation of the genomic evolution of HER2-positive breast cancer following progression on dual HER2-targetted therapy with Trastuzumab and Tucatinib [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-04-06.