Sensitive Next-generation Sequencing Research Articles

Automation of customizable library preparation for next-generation sequencing into an open microfluidic platform

Next-generation sequencing (NGS) is becoming more relevant for medical diagnostics, especially for using cell-free DNA to monitor response to therapy in cancer management, as high sensitivity of NGS enables detection of rare events. Sequencing Library preparation is a time-consuming and complex process, and large-scale liquid handlers are often used for automation. However, for smaller labs and low-to-medium throughput samples, these liquid handlers are expensive and need experts for handling. This work presents a proof-of-concept for library preparation on a commercially available and open lab-on-a-chip platform, which provides an alternative automation for low-to-medium throughput requirements. It covers common library preparation steps optimized to a microfluidic environment that include customizable PCR for target enrichment, end-repair, adapter ligation, nucleic acid purification via magnetic beads, and an integrated quantification step. The functionality of the cartridge is demonstrated with reference cfDNA containing different allelic frequencies of seven known mutations. Processing the samples in the cartridge reveals highly comparable results to manual processing (Pearson r = 0.94) based on amplicon sequencing. Summarized, the proposed automated lab-on-a-chip workflow for customizable library preparation could further pave the way for NGS to evolve from a technology used for research purposes to one that is applied in routine cancer management.

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.

Study on the value of second‐generation sequencing technology in the clinical diagnosis of osteoarticular brucellosis

AbstractTo assess the value of next‐generation sequencing (NGS) technology in the diagnosis of osteoarticular brucellosis pathogenesis. Fifty eight patients admitted to the Department of Orthopaedics, Hebei Provincial Chest Hospital from January 2021 to January 2023 were retrospectively analyzed, and the patients were classified into 48 cases in the osteoarticular brucellosis group and 10 cases in the nonosteoarticular brucellosis group according to the final clinical diagnosis. All patients underwent serum agglutination test (SAT), CT‐guided puncture or surgical sampling of lesions for bacteriological culture and NGS after admission. The diagnostic efficacy of these three methods for osteoarticular brucellosis was compared using the final clinical diagnosis as the reference standard. Among the 58 patients with suspected osteoarticular brucellosis, 40 cases (68.97%) were positive by NGS, 33 cases (56.89%) by SAT and 10 cases (17.24%) by culture, and the differences were statistically significant (p < 0.05). Using the final clinical diagnosis as a criterion, the sensitivity of NGS, SAT, and culture for the detection of osteoarticular brucellosis was 83.33%, 62.50%, and 20.83%, respectively, the specificity was 100.00%, 70.00%, and 100.00%, the diagnostic accuracy was 86.20%, 63.79%, and 34.49%, and the κ values were 0.799, 0.590, and 0.504, respectively. NGS has a high pathogen detection rate and sensitivity in the pathogenetic diagnosis of patients with osteoarticular brucellosis and can provide clinical guidance for the diagnosis and management of patients with osteoarticular brucellosis.

The Disassociation of A3G-Related HIV-1 cDNA G-to-A Hypermutation to Viral Infectivity.

APOBEC3G (A3G) restricts HIV-1 replication primarily by reducing viral cDNA and inducing G-to-A hypermutations in viral cDNA. HIV-1 encodes virion infectivity factor (Vif) to counteract A3G primarily by excluding A3G viral encapsidation. Even though the Vif-induced exclusion is robust, studies suggest that A3G is still detectable in the virion. The impact of encapsidated A3G in the HIV-1 replication is unclear. Using a highly sensitive next-generation sequencing (NGS)-based G-to-A hypermutation detecting assay, we found that wild-type HIV-1 produced from A3G-expressing T-cells induced higher G-to-A hypermutation frequency in viral cDNA than HIV-1 from non-A3G-expressing T-cells. Interestingly, although the virus produced from A3G-expressing T-cells induced higher hypermutation frequency, there was no significant difference in viral infectivity, revealing a disassociation of cDNA G-to-A hypermutation to viral infectivity. We also measured G-to-A hypermutation in the viral RNA genome. Surprisingly, our data showed that hypermutation frequency in the viral RNA genome was significantly lower than in the integrated DNA, suggesting a mechanism exists to preferentially select intact genomic RNA for viral packing. This study revealed a new insight into the mechanism of HIV-1 counteracting A3G antiviral function and might lay a foundation for new antiviral strategies.

Clonotypic VDJ Rearrangements in Mixed Phenotype Acute Leukemia can be Successfully Utilized to Track Minimal Residual Disease.

Multiplex polymerase chain reaction (PCR) and next-generation sequencing (NGS) can both be used to identify a neoplastic clonotype by targeting CDR3 and assessing rearrangements in IgH, IgK, IgL, TCR-β, and TCR-gamma loci. The clonotypic sequence can be robustly used to track minimal residual disease (MRD). The ability to track MRD by NGS in mixed phenotype acute leukemia (MPAL) is unknown and warrants investigation. Institutional Review Board (IRB) approval was obtained. Central Moffitt Cancer Center (MCC) database was searched to locate any patients with MPAL from over 600,000 entries. Patient charts were manually curated to identify those with clonoSEQ data, and clinical data was procured from the electronic medical record (EMR). Twenty-nine patients with MPAL were identified. Only 2 patients with clonoSEQ testing were found. Both demonstrated a B/myeloid phenotype, and both were bilineal. NGS (clonoSEQ) identified 4 dominant (IGH) (patient A; 8/2019) and 2 dominant sequences (patient B; 10/2019), respectively. In both patients, clonoSEQ testing successfully tracked minimal residual disease and mirrored clinical disease burden. This report is the first to confirm the utility of NGS-based MRD tracking in patients with MPAL and shows increased sensitivity of NGS over MRD flow cytometry.

Abstract PS07-09: Prenatal BRCA1 epimutations is a major cause of triple-negative breast cancer

Abstract Background: Low level normal cell BRCA1 epimutations have been associated with an increased risk of triple-negative breast cancer (TNBC). However, the fraction of TNBCs that may have BRCA1 epimutations as their underlying cause is unknown. Neither are the time of occurrence and the potential inheritance pattern of BRCA1 epimutations established. Methods: To address these questions, we analyzed BRCA1 methylation status in breast cancer tissue and matched white blood cells (WBC) from 411 patients with primary breast cancer, including 66 TNBCs. Samples were analyzed by a highly sensitive next-generation sequencing (NGS) assay on an Illumina MiSeq sequencer, allowing allele-resolved methylation assessment. Further, to assess the time of origin and the characteristics of normal cell BRCA1 methylation, we analyzed umbilical cord samples from 1260 newborn girls and 200 newborn boys.To assess potential Mendelian heritage, we analyzed BRCA1 methylation status in WBCs from 575 mothers and 531 fathers of newborn girls with (n = 102) and without (n = 473) WBC BRCA1 methylation. Results: We found concordant tumor and mosaic WBC BRCA1 epimutations in 10 out of 66 patients with TNBC and in four out of six patients with estrogen receptor (ER)-low expression (< 10%) of tumors (combined 14 out of 72; 19.4%, CI: 11.1-30.5). These exceeded the number of tumors harboring germline (n = 5) or somatic (n = 4) BRCA1 mutations. Notably, BRCA1 methylation and BRCA1 mutations were mutually exclusive. Contrasting the findings in TNBC and ER-low exprssion tumors, we found WBC and tumor BRCA1 methylation concordance in only three out of 221 patients with ER >10+% tumors and zero out of 116 patients with HER2 positive tumors. Intraindividually, BRCA1 epimutations affected the same allele in normal and tumor cells. Assessing BRCA1 methylation in umbilical cord WBCs from newborn girls, we found mosaic, predominantly monoallelic BRCA1 epimutations, with qualitative features similar to those in adults, in 113/1260 (9.0%) of individuals. We found no correlation between WBC BRCA1 methylation in newborns and methylation status in their mothers, fathers, or any parent. Notably, WBC BRCA1 methylation occurred at a significantly lower frequency in newborn boys ( 9/200; 4.5%) as compared to newborn girls (p = 0.038). Similarly, WBC BRCA1 methylation was found less common among fathers (16/531; 3.0%), as compared to mothers (46/575; 8.0%; p = 0.0003). Conclusions: Our findings suggest prenatal BRCA1 epimutations might be the underlying cause of around 20% of TNBC and low-ER expressing breast cancers. Such constitutional mosaic BRCA1 methylation likely arise through gender-related mechanisms in utero, independent of Mendelian inheritance. Citation Format: Per Lonning, Hans Petter Eikesdal, Elisabet Ognedal, Bjornar Gilje, Steinar Lundgren, Egil Blix, Helge Espelid, Jürgen Geisler, Stephanie Geisler, Emiel Janssen, Synnøve Yndestad, Laura Minsaas, Beryl Leirvaag, Reidun Lillestol, Stian Knappskog, Oleksii Nikolaienko. Prenatal BRCA1 epimutations is a major cause of triple-negative breast cancer [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 PS07-09.

Long-Term Follow-Up of Patients With Follicular Lymphoma Using Next Generation Sequencing to Detect Minimal Residual Disease

BackgroundFollicular lymphoma (FL) is a highly treatable, indolent non-Hodgkin lymphoma. Although FL is considered incurable, a patient without progression of disease by 24 months after treatment is predicted to have a survival consistent with persons without lymphoma. Using a sensitive assessment of minimal residual disease (MRD), we tested the hypothesis that MRD monitoring can predict long term remissions. MethodsUnselected patients who were in a clinical remission for at least 24 months after their last treatment were enrolled and monitored prospectively for MRD detectability using a sensitive next-generation sequencing assay (clonoSEQ, Adaptive Biotechnologies, Seattle, WA). ResultsForty-seven consecutive patients were monitored. We evaluated the MRD thresholds 10−4, 10−5, and 10−6 for the ability to predict long-term remissions in this cohort and determined that undetectable disease at 10−6 was the best predictor with a specificity and negative predictive value (NPV) of 70% and 100%, respectively. While 3 patients exhibited clinical disease progression during the course of the study, none of the 31 patients with persistent MRD undetectability at 10−6 experienced relapse. ConclusionsA significant proportion (31/47; 66.0%) of FL patients in clinical remission after ≥24 months following last therapy were undetectable at 10−6 by a sensitive assay of MRD. The threshold of sensitivity was 100%, specificity 70%, with a PPV of 19%, but a NPV of 100%. Although longer follow-up is needed for confirmation, many of these patients may continue to have durable complete remissions.

Evaluation of next-generation sequencing versus next-generation flow cytometry for minimal-residual-disease detection in Chinese patients with multiple myeloma

PurposeTo evaluate the efficacy of next-generation sequencing (NGS) in minimal-residual-disease (MRD) monitoring in Chinese patients with multiple myeloma (MM).MethodsThis study analyzed 60 Chinese MM patients. During MRD monitoring in these patients’ post-therapy, clonal immunoglobulin heavy chain (IGH) rearrangements were detected via NGS using LymphoTrack assays. MRD monitoring was performed using NGS or next-generation flow cytometry (NGF), and the results were compared. Additionally, the sensitivity and reproducibility of the NGS method were assessed.ResultsThe MRD detection range of the NGS method was 10–6–10–1, which suggested good linearity, with a Pearson correlation coefficient of 0.985 and a limit of detection of 10–6. Intra- and inter-assay reproducibility analyses showed that NGS exhibited 100% reproducibility with low variability in clonal cells. At diagnosis, unique clones were found in 42 patients (70.0%) with clonal IGH rearrangements, which were used as clonality markers for MRD monitoring post-therapy. Comparison of NGS and NGF for MRD monitoring showed 79.1% concordance. No samples that tested MRD-positive via NGF were found negative via NGS, indicating the higher sensitivity of NGS. MRD could be detected using NGS in 6 of 7 samples before autologous hematopoietic stem-cell transplantation, and 5 of them tested negative post-transplantation. In contrast, the NGF method could detect MRD in only 1 sample pre-transplantation.ConclusionCompared with NGF, NGS exhibits higher sensitivity and reproducibility in MRD detection and can be an effective strategy for MRD monitoring in Chinese MM patients.

The utility of liquid biopsy in clinical genetic diagnosis of cancer and monogenic mosaic disorders.

Liquid biopsy for minimally invasive diagnosis and monitoring of cancer patients is progressing toward routine clinical practice. With the implementation of highly sensitive next-generation sequencing (NGS) based assays for the analysis of cfDNA, however, consideration of the utility of liquid biopsy for clinical genetic testing is critical. While the focus of liquid biopsy for cancer diagnosis is the detection of circulating tumor DNA (ctDNA) as a fraction of total cell-free DNA (cfDNA), cfDNA analysis reveals both somatic mosaic tumor and germline variants and clonal hematopoiesis. Here we outline advantages and limitations of mosaic and germline variant detection as well as the impact of clonal hematopoiesis on liquid biopsy in cancer diagnosis. We also evaluate the potential of cfDNA analysis for the molecular diagnosis of monogenic mosaic disorders.

Development and validation of a next-generation sequencing assay with open-access analysis software for detecting resistance-associated mutations in CMV.

Cytomegalovirus (CMV) resistance testing by targeted next-generation sequencing (NGS) allows for the simultaneous analysis of multiple genes. We developed and validated an amplicon-based Ion Torrent NGS assay to detect CMV resistance mutations in UL27, UL54, UL56, and UL97 and compared the results to standard Sanger sequencing. NGS primers were designed to generate 83 overlapping amplicons of four CMV genes (~10 kb encompassing 138 mutation sites). An open-access software plugin was developed to perform read alignment, call variants, and interpret drug resistance. Plasmids were tested to determine NGS error rate and minor variant limit of detection. NGS limit of detection was determined using the CMV WHO International Standard and quantified clinical specimens. Reproducibility was also assessed. After establishing quality control metrics, 185 patient specimens previously tested using Sanger were reanalyzed by NGS. The NGS assay had a low error rate (<0.05%) and high accuracy (95%) for detecting CMV-associated resistance mutations present at ≥5% in contrived mixed populations. Mutation sites were reproducibly sequenced with 40× coverage when plasma viral loads were ≥2.6 log IU/mL. NGS detected the same resistance-associated mutations identified by Sanger in 68/69 (98.6%) specimens. In 16 specimens, NGS detected 18 resistance mutations that Sanger failed to detect; 14 were low-frequency variants (<20%), and six would have changed the drug resistance interpretation. The NGS assay showed excellent agreement with Sanger and generated high-quality sequence from low viral load specimens. Additionally, the higher resolution and analytic sensitivity of NGS potentially enables earlier detection of antiviral resistance.

Concordance of Next-Generation Sequencing and Multiparametric Flow Cytometry Methods for Detecting Measurable Residual Disease in Adult Acute Lymphoblastic Leukemia: Optimizing Prediction of Clinical Outcomes From a Single-Center Study

IntroductionDetection of measurable residual disease (MRD) in adults with acute lymphoblastic leukemia (ALL) is a vital biomarker in risk prediction and treatment selection. Next-generation sequencing (NGS) offers greater sensitivity relative to multiparametric flow cytometry (MFC) and may be a better predictive tool for identifying ALL patients at risk of relapse. Patients and MethodsThis single-center retrospective study compares MRD detection by NGS versus MFC in 52 adult B- and T-ALL patients treated at our institution between 2018 and 2023. Pretreatment bone marrow samples were used for assay calibration, while post-treatment MRD assessment was completed up to 4.5 months after the first complete remission (CR1) using an MRD cutoff of 10−6 for distinguishing relapse risk. ResultsThe 2-year cumulative incidence of relapse (CIR) among patients who were MRD positive using MFC and NGS was 39.5% and 46.2%, respectively. Unlike MFC, post-CR1 MRD positivity with NGS significantly predicted CIR (HR = 9.47, P = .028). In patients who were MRD negative by MFC, low levels of MRD detected by NGS distinguished patients at high risk of relapse (HR 10.3, P = .026, 2-year CIR 51.6%). ConclusionOur data suggests that assessment of post-CR1 MRD using a highly sensitive NGS assay can identify ALL patients undergoing frontline therapy at increased risk of relapse and guide the use of adjuvant therapy.

Minimally Invasive Assessment of Measurable Residual Disease (MRD) in Multiple Myeloma (MM)

BACKGROUND MRD is an established test in MM clinical trials but not in routine practice. A hindrance to its wider adoption is invasive bone marrow (BM) aspirates. Thus, the possibility of assessing MRD using minimally invasive methods could be paramount to facilitate its transition from clinical trials to routine practice, but also to improve patients' (pts) quality of life and to generate more meaningful MRD results based on periodic assessments instead of limited time points. However, the methods developed for MRD evaluation in BM are less sensitive when used in peripheral blood (PB). AIM Investigate the complementarity and prognostic value of new multimodal minimally invasive MRD assessment in MM. METHODS This study included 243 transplant eligible and ineligible pts who were on maintenance or observation at the time of MRD assessment in PETHEMA/GEM clinical trials. In all cases, MRD was evaluated in PB using BloodFlow, which is a highly sensitive method (10 -7) that combines immunomagnetic enrichment with next-generation flow (NGF) cytometry. Periodic assessment of MRD in peripheral blood (PB) using BloodFlow was performed every six months. In 27 patients, MRD was further investigated in cfDNA using CloneSight, a highly sensitive (10 -4) next-generation sequencing method based on pt-specific mini-panels of multiplexed amplicons covering somatic mutations identified at diagnosis. In 169 of the 243 patients, MRD was analyzed in the serum using Quantitative Immunoprecipitation Mass Spectrometry (QIP-MS) with anti-IgG/A/M, total κ and λ beads. RESULTS BloodFlow was performed in a total of 867 PB samples, out of which 77 (9%) were MRD positive. The median number of CTCs/µL was 0.016 (range, 0.0003 - 0.29). In 506 of the 867 assessments, MRD was simultaneously analyzed in the BM using NGF. The concordance between BloodFlow in PB and NGF in BM was 78.9% (70.4% double negative and 8.5% double positive MRD results). The frequency of BloodFlow-/NGF+ and BloodFlow+/NGF- discordant assessments was 20.3% and 0.8%, respectively. Serial assessment of MRD in PB using BloodFlow showed that 221 of the 243 (91%) pts had sustained MRD negativity and 13 (5%) were persistently positive. MRD conversions from negative to positive were noted in 9 (4%) pts. The landmark median PFS from the latest MRD assessment was not reached (NR) vs 3 months in negative vs positive patients (HR: 0.09, P &amp;lt; .001). Of note, only 11 of the 221 (5%) MRD negative pts relapsed thus far. Of the 27 pts in whom MRD was simultaneously analyzed in cfDNA using CloneSight, five were MRD positive. The landmark median PFS from the latest MRD assessment was NR vs 5 months in negative vs positive pts (HR: 0.23, P = .057). The number of patients with BloodFlow-/CloneSight-, BloodFlow+/CloneSight-, BloodFlow-/CloneSight+ and BloodFlow+/CloneSight+ results were 17, 5, 2 and 3. The landmark median PFS from the latest MRD assessment in patients with double negative MRD vs positive by either method was NR vs 8 months (HR: 0.20, P = .056). Of note, only 2 of the 17 double negative MRD patients relapsed thus far. Of the 169 pts in whom MRD was simultaneously analyzed in serum using QIP-MS, 37 (22%) were MRD positive. The landmark median PFS from the latest MRD assessment was NR in negative and positive patients (HR: 0.13, P &amp;lt; .001). The number of patients with BloodFlow-/QIPMS-, BloodFlow+/QIPMS-, BloodFlow-/QIPMS+ and BloodFlow+/QIPMS+ results were 130, 2, 26 and 11. The landmark median PFS from the latest MRD assessment in patients with double negative MRD vs positive by either method was NR and 8 months, respectively (HR: 0.07, P &amp;lt; .001). Of note, only 3 of the 130 (2%) double negative MRD patients relapsed thus far. The negative predictive value (NPV) of a double negative MRD detection in PB/serum using BloodFlow/QIP-MS and MRD negativity in BM using NGF was 86%. CONCLUSIONS This is the first study of multimodal minimally invasive MRD assessment in MM. Our results show that BloodFlow, CloneSight and QIP-MS are empowered to detect MRD with high sensitivity in PB, cfDNA and serum. The presence of CTCs, mutations and M-component was systematically associated with dismal PFS. The complementarity between these methods achieved a high NPV of MRD negativity in BM, and enabled the identification of multimodal MRD negative pts with very low risk of relapse. Thus, this study paves the way towards minimally invasive MRD assessment in MM pts on maintenance or observation.

Ultrasensitive NGS MRD assessment in Ph+ ALL: Prognostic impact and correlation with RT-PCR for BCR::ABL1.

Reverse transcription polymerase chain reaction (RT-PCR) for BCR::ABL1 is the most common and widely accepted method of measurable residual disease (MRD) assessment in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL); however, RT-PCR may not be an optimal measure of MRD in many cases of Ph+ ALL. We evaluated the clinical impact of a highly sensitive next-generation sequencing (NGS) MRD assay (sensitivity of 10-6 ) and its correlation with RT-PCR for BCR::ABL1 in patients with Ph+ ALL. Overall, 32% of patients had a discordance between MRD assessment by RT-PCR and NGS, and 31% of patients who achieved NGS MRD negativity were PCR+ at the same timepoint. Among eight patients with long-term detectable BCR::ABL1 by PCR, six were PCR+/NGS-. These patients generally had stable PCR levels that persisted despite therapeutic interventions, and none subsequently relapsed; in contrast, patients who were PCR+/NGS+ had more variable PCR values that responded to therapeutic intervention. In a separate cohort of prospectively collected clinical samples, 11 of 65 patients (17%) with Ph+ ALL who achieved NGS MRD negativity had detectable BCR::ABL1 by PCR, and none of these patients relapsed. Relapse-free survival and overall survival were similar in patients who were PCR+/NGS- and PCR-/NGS-, suggesting that PCR for BCR::ABL1 did not provide additional prognostic information in patients who achieved NGS MRD negativity. NGS-based assessment of MRD is prognostic in Ph+ ALL and identifies patients with low-level detectable BCR::ABL1 who are unlikely to relapse nor to benefit from therapeutic interventions.

Evaluation of a commercial NGS service for detection of bacterial and fungal pathogens in infectious ulcerative keratitis.

To compare results from a commercial next-generation sequencing (NGS) service to corneal cytology and culture for identification of causative organisms in veterinary patients presenting for infectious ulcerative keratitis (IUK). Swabs for corneal aerobic and fungal cultures and DNA swabs for NGS were submitted for canine and equine normal controls (n=11 and n=4, respectively) and IUK patients (n=22 and n=8, respectively) for which microbrush cytology specimens confirmed the presence of infectious organisms. The sensitivity of the NGS results was compared with bacterial and fungal culture results. Concordance between the NGS and culture results was determined. The NGS results were positive for bacterial and fungal organisms in 5 and 1 normal and 18 and 1 IUK cases, respectively. Bacterial and fungal cultures were positive for 7 and 2 normal and 20 and 5 IUK cases, respectively. Sensitivity of NGS was 82.14% (95% confidence interval (CI), 63.11% to 93.94%) and specificity was 76.47% (95% CI, 50.10% to 93.19%). Concordance (complete and partial) between identified bacterial and fungal organisms was found in 79% and 100% of cases, respectively. NGS identified organisms in 3 culture-negative IUK samples. A commercial NGS service may be useful in the identification of causative agents in IUK cases with a sensitivity greater than the sensitivity previously reported for aerobic culture. Further testing is needed to determine the clinical significance of additional organisms isolated by NGS from infected cases, as well as organisms isolated from normal corneas.

Insertions and deletions in the hypervariable region of the hepatitis E virus genome in individuals with acute and chronic infection.

Hepatitis E virus is a major cause of acute hepatitis worldwide and can progress to chronicity in immunocompromised individuals. Various virus-host recombination events have been reported in the hypervariable region of the hepatitis E virus genome, but the patterns of assembly and selection remain unclear. To gain further insight into viral evolution, we assessed the presence of low abundance variants in 16 samples from individuals with acute or chronic infection using a targeted next-generation sequencing approach. In seven samples, different variants with insertions and/or deletions were identified. Among them, eight insertions originating either from human genes or from the hepatitis E virus genome. Five different deletions could be identified. The amino acid composition of sequences with insertions showed a higher frequency of lysine and a lower abundance of proline, and additionally acetylation and ubiquitination sites were more frequent than in hepatitis E virus wild-type sequences. These findings suggest that the nucleotide composition of insertions and sites for post-translational modification may contribute to recombination events. Although the impact of low-level hepatitis E virus variants is uncertain, our results highlight the importance of a highly sensitive next-generation sequencing approach to capture the full diversity of hypervariable region.

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.

Mitochondrial DNA Enrichment for Sensitive Next-Generation Sequencing.

Mitochondrial DNA (mtDNA) encodes components essential for cellular respiration. Low levels of point mutations and deletions accumulate in mtDNA during normal aging. However, improper maintenance of mtDNA results in mitochondrial diseases, stemming from progressive loss of mitochondrial function through the accelerated formation of deletions and mutations in mtDNA. To better understand the molecular mechanisms underlying the creation and propagation of mtDNA deletions, we developed the LostArc next-generation DNA sequencing pipeline to detect and quantify rare mtDNA species in small tissue samples. LostArc procedures are designed to minimize PCR amplification of mtDNA and instead achieve enrichment of mtDNA by selective destruction of nuclear DNA. This approach leads to cost-effective, high-depth sequencing of mtDNA with a sensitivity sufficient to identify one mtDNA deletion per million mtDNA circles. Here, we describe detailed protocols for isolation of genomic DNA from mouse tissues, enrichment of mtDNA through enzymatic destruction of linear nuclear DNA, and preparation of libraries for unbiased next-generation sequencing of mtDNA.

Added Value of Next Generation over Sanger Sequencing in Kenyan Youth with Extensive HIV-1 Drug Resistance.

HIV-1 drug resistance testing in children and adolescents in low-resource settings is both important and challenging. New (more sensitive) drug resistance testing technologies may improve clinical care, but evaluation of their added value is limited. We assessed the potential added value of using next-generation sequencing (NGS) over Sanger sequencing for detecting nucleoside reverse transcriptase inhibitor (NRTI) and nonnucleoside reverse transcriptase inhibitor (NNRTI) drug resistance mutations (DRMs). Participants included 132 treatment-experienced Kenyan children and adolescents with diverse HIV-1 subtypes and with already high levels of drug resistance detected by Sanger sequencing. We examined overall and DRM-specific resistance and its predicted impact on antiretroviral therapy and evaluated the discrepancy between Sanger sequencing and six NGS thresholds (1%, 2%, 5%, 10%, 15%, and 20%). Depending on the NGS threshold, agreement between the two technologies was 62% to 88% for any DRM, 83% to 92% for NRTI DRMs, and 73% to 94% for NNRTI DRMs, with more DRMs detected at low NGS thresholds. NGS identified 96% to 100% of DRMs detected by Sanger sequencing, while Sanger identified 83% to 99% of DRMs detected by NGS. Higher discrepancy between technologies was associated with higher DRM prevalence. Even in this resistance-saturated cohort, 12% of participants had higher, potentially clinically relevant predicted resistance detected only by NGS. These findings, in a young, vulnerable Kenyan population with diverse HIV-1 subtypes and already high resistance levels, suggest potential benefits of more sensitive NGS over existing technology. Good agreement between technologies at high NGS thresholds supports their interchangeable use; however, the significance of DRMs identified at lower thresholds to patient care should be explored further. IMPORTANCE HIV-1 drug resistance in children and adolescents remains a significant problem in countries facing the highest burden of the HIV epidemic. Surveillance of HIV-1 drug resistance in children and adolescents is an important public health strategy, particularly in resource-limited settings, and yet, it is limited due mostly to cost and infrastructure constraints. Whether newer and more sensitive next-generation sequencing (NGS) adds substantial value beyond traditional Sanger sequencing in detecting HIV-1 drug resistance in real life settings remains an open and debatable question. In this paper, we attempt to address this issue by performing a comprehensive comparison of drug resistance identified by Sanger sequencing and six NGS thresholds. We conducted this study in a well-characterized, vulnerable cohort of children and adolescents living with diverse HIV-1 subtypes in Kenya and, importantly, failing antiretroviral therapy (ART) with already extensive drug resistance. Our findings suggest a potential added value of NGS over Sanger even in this unique cohort.

Development of a Standardized, DNA-Based Next Generation Sequencing Assay for Assessment of Measurable Residual Disease (MRD) in Acute Myeloid Leukemia (AML) As the Primary Endpoint in the Agility Study

Background:NPM1 mutations (NPM1m) are a well-characterized biomarker for minimal residual disease (MRD) detection in acute myeloid leukemia (AML). Based on our retrospective meta-analysis across multiple trials and prior published studies, we are evaluating the utility of MRD complete response as a surrogate biomarker for clinical benefit in the AGILITY study (NCT05020665) to support a potential accelerated approval. Currently, measurement of NPM1m RNA transcripts (relative to ABL1) using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is considered the gold standard for monitoring of MRD; however, DNA-based next generation sequencing (NGS) provides several logistical and technical advantages over RT-qPCR in a global registrational trial setting. NGS allows for detection of all NPM1m variant alleles in a single assay (more closely approximating the variant allelic fraction [VAF] from leukemic cells vs NPM1m transcripts). Furthermore, longer stability of DNA during transit alleviates logistical challenges related to sample processing across different labs and transport from sites worldwide to a central testing site. Purpose: To develop a standardized and highly sensitive DNA-based NGS assay for detection of MRD in NPM1m AML patients at the end of 2 cycles of chemotherapy in the AGILITY study and to assess its performance against an established RT-qPCR method. Methods: Assay performance was assessed by serially diluting NPM1 synthetic DNA or NPM1m genomic DNA (gDNA) extracted from OCI/AML3 cells in a background of wild-type bone marrow (BM) gDNA (range: 0.008%-0.0005% VAF) using a fixed sample input of 400,000 gene copies. DNA sequencing (median read depth: 5.5×106) analysis based on the Inivata MRD platform was performed on the Illumina NextSeq 550 instrument using a proprietary bioinformatic pipeline. A set of follow-up AML samples consisting of peripheral blood (N = 31) and BM (N = 55) from patients enrolled in the UK NCRI AML Study Group AML-19 trial (ISRCTN 2014-002195-90) were tested for comparison of MRD status by NGS with an orthogonal NPM1 RT-qPCR method. This sample cohort included both negative (n = 16) and positive (n = 70) samples as defined by RT-qPCR analysis. To assess the level of agreement between both platforms, we use Pearson's correlation statistics including 95% CIs. Assay performance was evaluated by determining the area under the receiver operating characteristic curve (ROCAUC). RT-qPCR was performed as previously described (Ivey A. N Engl J Med. 2016). NGS results are expressed as percent variant allele frequency (%VAF) and RT-qPCR results as NPM1m transcript copies per 10e4ABL transcripts (normalized copy number [NCN]). Results: The NPM1m NGS assay demonstrated high sensitivity and specificity with a limit of detection with 95% confidence (LoD95) of 0.002% VAF when using serially diluted NPM1 synthetic DNA or NPM1m gDNA extracted from OCI/AML3 cells. In the AML19 set, MRD was detectable in 52/70 (74.3%) samples by both platforms. The level of MRD detected by NGS varied between 0.00025%-46% VAF (median: 0.0042%) and by RT-qPCR between 0.01-1.65x105 NCN (median: 8.03), showing a statistically significant correlation when considering all positive samples (R = 0.84, P < 9.8e-15). Despite this agreement, in 18/70 (25.7%) samples NPM1m was not detected by NGS but detected by RT-qPCR, with NCN between 0.03 and 20.5 (median: 0.37), confirming the expected higher sensitivity of RT-qPCR. Conversely, MRD was detected in 5/16 samples by NGS but not detected by RT-qPCR (VAFs were < technical LoD95 in all cases). The NGS assay could reliably detect MRD positivity in almost all samples with an NCN > 10 copies (23/24 samples, 95.8%) and there was a statistically significant correlation (R = 0.78, P < 1.4e-5). Using a cutoff of > 10 copies, ROC analysis was performed, again demonstrating good concordance (AUC: 91.9%) and identifying an optimal cutoff of 0.003% VAF (specificity = 85.5%, sensitivity = 87.5%). Conclusion: Our DNA-based NGS-MRD assay demonstrates high sensitivity and specificity with a technical LoD95 in serial dilution of 0.002% VAF. It is sufficiently robust to reliably identify MRD positive samples above an RT-qPCR threshold of 10 copies / 10e4ABL (ie, 0.01% NPM1/ABL ratio) and notably, this is tenfold below the provisional European LeukemiaNet definition for high-level MRD of > 0.1%.

Identification of microbial taxa present in Ctenocephalides felis (cat flea) reveals widespread co-infection and associations with vector phylogeny

BackgroundCtenocephalides felis, the cat flea, is the most common ectoparasite of cats and dogs worldwide. As a cause of flea allergy dermatitis and a vector for two genera of zoonotic pathogens (Bartonella and Rickettsia spp.), the effect of the C. felis microbiome on pathogen transmission and vector survival is of substantial medical importance to both human and veterinary medicine. The aim of this study was to assay the pathogenic and commensal eubacterial microbial communities of individual C. felis from multiple geographic locations and analyze these findings by location, qPCR pathogen prevalence, and flea genetic diversity.Methods16S Next Generation Sequencing (NGS) was utilized to sequence the microbiome of fleas collected from free-roaming cats, and the cox1 gene was used for flea phylogenetic analysis. NGS data were analyzed for 168 individual fleas from seven locations within the US and UK. Given inconsistency in the genera historically reported to constitute the C. felis microbiome, we utilized the decontam prevalence method followed by literature review to separate contaminants from true microbiome members.ResultsNGS identified a single dominant and cosmopolitan amplicon sequence variant (ASV) from Rickettsia and Wolbachia while identifying one dominant Bartonella clarridgeiae and one dominant Bartonella henselae/Bartonella koehlerae ASV. Multiple less common ASVs from these genera were detected within restricted geographical ranges. Co-detection of two or more genera (Bartonella, Rickettsia, and/or Wolbachia) or multiple ASVs from a single genus in a single flea was common. Achromobacter, Peptoniphilus, and Rhodococcus were identified as additional candidate members of the C. felis microbiome on the basis of decontam analysis and literature review. Ctenocephalides felis phylogenetic diversity as assessed by the cox1 gene fell within currently characterized clades while identifying seven novel haplotypes. NGS sensitivity and specificity for Bartonella and Rickettsia spp. DNA detection were compared to targeted qPCR.ConclusionsOur findings confirm the widespread coinfection of fleas with multiple bacterial genera and strains, proposing three additional microbiome members. The presence of minor Bartonella, Rickettsia, and Wolbachia ASVs was found to vary by location and flea haplotype. These findings have important implications for flea-borne pathogen transmission and control.Graphical