Next-generation Sequencing Workflow Research Articles

Use of synthetic circular RNA spike-ins (SynCRS) for normalization of circular RNA sequencing data.

High-throughput RNA sequencing enables the quantification of transcript abundance and the identification of novel transcripts in biological samples. These include circular RNAs (circRNAs), a family of alternatively spliced RNA molecules that form a continuous loop. However, quantification and comparison of circRNAs between RNA sequencing libraries remain challenging due to confounding errors introduced during exonuclease digestion, library preparation and RNA sequencing itself. Here we describe a set of synthetic circRNA spike-ins-termed 'SynCRS'-that can be added directly to purified RNA samples before exonuclease digestion and library preparation. SynCRS, introduced either individually or in combinations of varying size and abundance, can be integrated into all next-generation sequencing workflows and, critically, facilitate the quantitative calibration of circRNA transcript abundance between samples, tissue types, species and laboratories. Our step-by-step protocol details the generation of SynCRS and guides users on the stoichiometry of SynCRS spike-in to RNA samples, followed by the bioinformatic steps required to facilitate quantitative comparisons of circRNAs between libraries. The laboratory steps to produce the SynCRS require an additional 3 d on top of the high throughput circRNA sequencing and bioinformatics. The protocol is suitable for users with basic experience in molecular biology and bioinformatics.

Cancer associated variant enrichment CAVE, a gene agnostic approach to identify low burden variants in chronic lymphocytic leukemia

Intratumoral heterogeneity is an important clinical challenge because low burden clones expressing specific genetic alterations drive therapeutic resistance mechanisms. We have developed CAVE (cancer-associated variant enrichment), a gene-agnostic computational tool to identify specific enrichment of low-burden cancer driver variants in next-generation sequencing (NGS) data. For this study, CAVE was applied to TP53 in chronic lymphocytic leukemia (CLL) as a cancer model. Indeed, as TP53 mutations are part of treatment decision-making algorithms and low-burden variants are frequent, there is a need to distinguish true variants from background noise. Recommendations have been published for reliable calling of low-VAF variants of TP53 in CLL and the assessment of the background noise for each platform is essential for the quality of the testing. CAVE is able to detect specific enrichment of low-burden variants starting at variant allele frequencies (VAFs) as low as 0.3%. In silico TP53 dependent and independent analyses confirmed the true driver nature of all these variants. Orthogonal validation using either ddPCR or NGS analyses of follow-up samples confirmed variant identification. CAVE can be easily deployed in any cancer-related NGS workflow to detect the enrichment of low-burden variants of clinical interest.

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).

Primary immune regulatory disorders (PIRD): expanding the mutation spectrum in Turkey and identification of sixteen novel variants

Human Inborn Errors of Immunity (IEIs) encompass a clinically and genetically heterogeneous group of disorders, ranging from mild cases to severe, life-threatening types. Among these, Primary Immune Regulatory Disorders (PIRDs) constitute a subset of IEIs characterized by diverse clinical phenotypes, prominently featuring severe atopy, autoimmunity, lymphoproliferation, hyperinflammation, autoinflammation, and susceptibility to malignancies. According to the latest report from the International Union of Immunological Societies (IUIS), PIRDs arise from mutations in various genes including LYST, RAB27A, AP3B1, AP3D1, PRF1, UNC13D, STX11, STXBP2, FAAP24, SLC7A7, RASGRP1, CD70, CTPS1, RLTPR, ITK, MAGT1, PRKCD, TNFRSF9, SH2DIA, XIAP, CD27 (TNFRSF7), FAS (TNFRSF6), FASLG (TNFSF6), CASP10, CASP8, FADD, LRBA, STAT3, AIRE, ITCH, ZAP70, TPP2, JAK1, PEPD, FOXP3, IL2RA, CTLA4, BACH2, IL2RB, DEF6, FERMT1, IL10, IL10RA, IL10RB, NFAT5, TGFB1, and RIPK1 genes. We designed a targeted next-generation sequencing (TNGS) workflow using the Ion AmpliSeq™ Primary Immune Deficiency Research Panel to sequence 264 genes associated with IEIs on the Ion S5™ Sequencer. In this study, we report the identification of 38 disease-causing variants, including 16 novel ones, detected in 40 patients across 15 distinct PIRD genes. The application of next-generation sequencing enabled rapid and precise diagnosis of patients with PIRDs.

Abstract 949: A single stranded DNA library preparation workflow used for hybridization capture based WES assay showed superior uniformity

Abstract Introduction: In many instances in Precision Oncology Medicine fragmented DNA from formalin-fixed paraffin-embedded (FFPE) samples is the only option. The majority of such degradedsamples need to be sequenced using next generation sequencing (NGS) [1]. Standardizedguidelines highlight that a minimum of 500 × coverage depth is required for tissue analyses [2].However, coverage isn’t the only metric required for a successful NGS analysis. Highly uniformDNA libraries allow clinical labs to generate more hits from their screens, saving both time andmoney across the course of diagnosis therefore ultimately benefits patient care. The commerciallyavailable single stranded DNA library prep kit tested in this study renders all input moleculessingle-stranded before ligation therefore it recovers template molecules containing DNA nicks andlabile lesions thus maximizing library complexity. Method: 10 solid tumor FFPE derived DNA samples went through three different NGS workflows:100ng of DNA input in an amplicon-based assay 325 gene panel (Assay A), 60ng input for a 500gene mechanical sheared-based double strand DNA library preparation with hybridization captureassay (~500 gene panel, Assay B), 50ng and 10ng input for a enzymatic shear-based singlestranded DNA library preparation with hybridization capture assay (WES with 1000+ enhancedgene content, Assay C). Data from assays A and C was processed with in-house bioinformaticpipeline, while Assay B’s data was processed with a commercially available bioinformatic pipelinethat was bundled with the assay. NGS QC metrics were compared across the three datasets. Results: This novel single stranded DNA library preparation with hybridization capture workflowshowed superior QC metrics compared to the other two workflows, especially on coverageuniformity and percent target above 100X, with as low as 10ng input. Conclusion: Employing a novel commercially available DNA library preparation workflow in anNGS assay, NeoGenomics was able to rescue poor quality samples that had previously failed tomeet our internal uniformity metric on other NGS assays and generate robust and reportableresults with as low as 10ng DNA input.[1] Nagahashi et al., J Surg Res. 2017 Dec; 220: 125-132.[2] Cappello et al., J Pers Med. 2022 May; 12(5): 750. Citation Format: Jieying Chu, Rachel Schell, Faqiang Wu, Michal Krawczyk, Daniel Whang, Segun Jung, Hyunjun Nam, Kenneth B. Thomas, Fernando J. Lopez-Diaz, Vincent Funari, Julie A. Mayer, Shashi Kulkarni, Jiannan Guo, Steven P. Lau-Rivera. A single stranded DNA library preparation workflow used for hybridization capture based WES assay showed superior uniformity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 949.

Abstract 5054: Optimization and evaluation of an FFPE dual extraction protocol for next-generation sequencing applications

Abstract Formalin-fixed paraffin-embedded (FFPE) biopsies are highly valuable and widely used tissue specimens for clinical diagnostics. However, obtaining sufficient and high-quality nucleic acid material from limited FFPE samples presents a challenge for downstream molecular analysis, such as next-generation sequencing (NGS). We present an optimized sequential extraction method that generates high-quality DNA and RNA from a single set of input tissues that is automatable and operation-friendly. This workflow performs well with reduced FFPE tissue input and efficiently supports various high-throughput clinical NGS applications. The DNA/RNA yield, quality, purity, and impacts on NGS assay performances were on par or better than an existing validated comparator extraction method. With comparable FFPE input, the new method demonstrated a superior extraction performance with significantly higher yield, quality, and purity. For DNA, NGS libraries were made with two different library preparation methods: a TA-ligation-based method and a single-strand-based method, followed by hybrid capturing and sequencing. The DNA from the new extraction method demonstrated a superior library conversion rate and improved target enrichment uniformity with both chemistries. This provides the potential of reduced input requirements, allowing very limited tissue, such as fine needle aspirate or core needle biopsy, for clinical NGS testing. The sequencing results were highly concordant between the existing and new extraction methods when the extracted DNA was subjected to a validated comprehensive genomic profiling (CGP) clinical test, demonstrating the quality and robustness of the extraction. For RNA, NGS libraries were made following the validated CGP clinical test with hybrid capturing, and the results were comparable between the two extraction methods. In conclusion, an FFPE extraction method was optimized to allow more clinical biopsy samples to be tested with different NGS workflows, providing a better diagnostic value for patient care. Citation Format: Rebecca Hong, Jieying Chu, Steven Hsiao, Daniel Whang, Steven P. Rivera, Heng Xie, Jiannan Guo. Optimization and evaluation of an FFPE dual extraction protocol for next-generation sequencing applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5054.

Abstract 317: Using the Biomek NGeniuS Next Generation Library Prep System to automate the Illumina TruSight Oncology 500 assay

Abstract Cancer researchers using next generation sequencing (NGS) need reliable sample preparation methodologies that generate high-quality, sequence-ready libraries from often-challenging samples. This process can be aided using automated liquid handling systems. The Biomek NGeniuS Next Generation Library Prep System from Beckman Coulter Life Sciences is a reliable, easy-to-use, purpose-built liquid handler for NGS library preparation ideal for laboratories requiring more flexibility and less hands-on time. The user-friendly software requires no programming skills, making it easy to set up and run. Biomek NGeniuS Next Generation Library Prep System’s hardware features include an on-deck thermal cycler, gripper, temperature-controlled reagent storage, and high-density tip storage which reduces user interventions and enables more walkaway time. An extensive and growing library of complimentary demonstrated NGS sample prep protocols allows the user to select from a variety of applications for NGS workflows. One such workflow is Illumina TruSight Oncology 500 assay, which represents a comprehensive NGS assay targeting the full coding regions of 523 genes implicated in the pathogenesis of solid tumors. Using enrichment-based library preparation techniques for use with formalin-fixed, paraffin-embedded (FFPE) samples, the Illumina TruSight Oncology 500 assay, when running the DNA Only workflow, can detect single nucleotide variants (SNVs), indels, amplifications, and multinucleotide variants (MNVs) in a single sequencing run. The Illumina TruSight Oncology 500 assay also detects immunotherapy biomarkers for tumor mutational burden (TMB) and microsatellite instability (MSI) in DNA samples. The Illumina TruSight Oncology 500 assay also offers a combined RNA/DNA workflow that also allows for the assessment of fusion events. In this poster, we show the utility of the Biomek NGeniuS Next Generation Library Prep System to perform the Illumina TruSight Oncology 500 assay, which saves researchers valuable hands-on time and offers a highly streamlined setup process for users new to liquid handling automation. Confidential - Company Proprietary Citation Format: Zach Smith. Using the Biomek NGeniuS Next Generation Library Prep System to automate the Illumina TruSight Oncology 500 assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 317.

Abstract 5035: Use of Streck nucleic acid BCT with plasma nucleic acid next-generation sequencing workflows

Abstract Introduction: Liquid biopsies, which interrogate body fluids in search of analytes released by cancer cells, are becoming increasingly common in clinical oncology. Foremost among the technologies used are those that employ nucleic acids, such as cell-free DNA (cfDNA) and cell-free RNA (cfRNA). Currently, many NGS-based tests perform near the limit of assay detection, making the identification of critical mutations, fusions, or expression patterns like finding a needle in a haystack. Further hindering successful assay development is the variability in analyte availability and detectability that can occur prior to analysis due to blood collection, transport, and storage. Here, we demonstrate that Nucleic Acid BCT stabilizes draw-time levels of plasma nucleic acids for downstream use with NGS-based assays. Methods: Blood from self-proclaimed healthy donors was drawn into EDTA and Nucleic Acid BCT and stored at ambient temperature for up to 7 days before plasma isolation with a general double-spin protocol and storage at -80 °C. Plasma nucleic acids were isolated using the MAGicBead cfDNA Kit (Zymo, cfDNA) or the QIAamp Circulating Nucleic Acid Kit (QIAGEN, cfRNA). Library prep for cfDNA was carried out with added synthetic mutant spike-in oligonucleotides using the Illumina TruSight Tumor 15 kit (Illumina). cfRNA library prep was done with the NEBNext Ultra II RNA Library Prep Kit for Illumina with upfront rRNA depletion (New England Biolabs). Sequencing was performed using an Illumina NextSeq 500 system, and all data were saved in and analyzed with Illumina BaseSpace applications. Results: When blood samples were stored in EDTA tubes for up to 7 days, spiked mutant alleles (EGFRL858R, EGFRdel19, KRASG12D, and PIK3CAE545K) were no longer detected, likely due to a dilution of the spiked DNA concentration following the release of genomic DNA containing wildtype alleles during white blood cell degradation. In contrast, equivalent blood samples stored in Nucleic Acid BCTs displayed mutant allele frequencies reflective of draw-time levels. Interrogation of plasma RNA expression patterns in samples stored in Nucleic Acid BCT versus EDTA revealed similar results, where differentially expressed plasma transcripts in blood samples stored in EDTA were found at high levels (>1,000 transcripts) versus donor-matched blood samples stored in Nucleic Acid BCT (<50 transcripts). Conclusions: Our data demonstrate that Nucleic Acid BCT maintains mutant allele frequencies and the plasma transcriptome profile of blood samples during ambient temperature storage. This provides precious sample integrity during whole blood storage and transport, offering laboratories and assay developers reduced preanalytical variability for NGS-based analysis of gene mutations, fusions, indels, and the plasma transcriptome. Nucleic Acid BCT is for Research Use Only. Not for use in diagnostic procedures. Citation Format: Nicholas M. George, Lisa Bartron. Use of Streck nucleic acid BCT with plasma nucleic acid next-generation sequencing workflows [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5035.

Subclonal Cancer Driver Mutations Are Prevalent in the Unresected Peritumoral Edema of Adult Diffuse Gliomas.

True2 is a next-generation sequencing workflow that facilitates unbiased discovery of somatic mutations across the full range of variant allele frequencies, which could help identify residual disease vulnerabilities for targeted adjuvant therapies.

Recommendations for laboratory workflow that better support centralised amalgamation of genomic variant data: findings from CanVIG-UK national molecular laboratory survey.

National and international amalgamation of genomic data offers opportunity for research and audit, including analyses enabling improved classification of variants of uncertain significance. Review of individual-level data from National Health Service (NHS) testing of cancer susceptibility genes (2002-2023) submitted to the National Disease Registration Service revealed heterogeneity across participating laboratories regarding (1) the structure, quality and completeness of submitted data, and (2) the ease with which that data could be assembled locally for submission. In May 2023, we undertook a closed online survey of 51 clinical scientists who provided consensus responses representing all 17 of 17 NHS molecular genetic laboratories in England and Wales which undertake NHS diagnostic analyses of cancer susceptibility genes. The survey included 18 questions relating to 'next-generation sequencing workflow' (11), 'variant classification' (3) and 'phenotypical context' (4). Widely differing processes were reported for transfer of variant data into their local LIMS (Laboratory Information Management System), for the formatting in which the variants are stored in the LIMS and which classes of variants are retained in the local LIMS. Differing local provisions and workflow for variant classifications were also reported, including the resources provided and the mechanisms by which classifications are stored. The survey responses illustrate heterogeneous laboratory workflow for preparation of genomic variant data from local LIMS for centralised submission. Workflow is often labour-intensive and inefficient, involving multiple manual steps which introduce opportunities for error. These survey findings and adoption of the concomitant recommendations may support improvement in laboratory dataflows, better facilitating submission of data for central amalgamation.

Multicenter evaluation of gut microbiome profiling by next-generation sequencing reveals major biases in partial-length metabarcoding approach

Next-generation sequencing workflows, using either metabarcoding or metagenomic approaches, have massively contributed to expanding knowledge of the human gut microbiota, but methodological bias compromises reproducibility across studies. Where these biases have been quantified within several comparative analyses on their own, none have measured inter-laboratory reproducibility using similar DNA material. Here, we designed a multicenter study involving seven participating laboratories dedicated to partial- (P1 to P5), full-length (P6) metabarcoding, or metagenomic profiling (MGP) using DNA from a mock microbial community or extracted from 10 fecal samples collected at two time points from five donors. Fecal material was collected, and the DNA was extracted according to the IHMS protocols. The mock and isolated DNA were then provided to the participating laboratories for sequencing. Following sequencing analysis according to the laboratories’ routine pipelines, relative taxonomic-count tables defined at the genus level were provided and analyzed. Large variations in alpha-diversity between laboratories, uncorrelated with sequencing depth, were detected among the profiles. Half of the genera identified by P1 were unique to this partner and two-thirds of the genera identified by MGP were not detected by P3. Analysis of beta-diversity revealed lower inter-individual variance than inter-laboratory variances. The taxonomic profiles of P5 and P6 were more similar to those of MGP than those obtained by P1, P2, P3, and P4. Reanalysis of the raw sequences obtained by partial-length metabarcoding profiling, using a single bioinformatic pipeline, harmonized the description of the bacterial profiles, which were more similar to each other, except for P3, and closer to the profiles obtained by MGP. This study highlights the major impact of the bioinformatics pipeline, and primarily the database used for taxonomic annotation. Laboratories need to benchmark and optimize their bioinformatic pipelines using standards to monitor their effectiveness in accurately detecting taxa present in gut microbiota.

From research genomics to clinical genetics: Insourcing next generation sequencing in a pediatric and women’s hospital

Abstract Introduction/Objective The Sidra Medicine Clinical Genomics Laboratory (CGL) offers rapid and cost-effective next- generation sequencing (NGS) solutions such as Whole Genome Sequencing (WGS) and Whole Exome Sequencing (WES) which support various research initiatives in Qatar. With the current worldwide trend in precision and personalized medicine, the Pathology Genetics (PG) division is required to provide in-house diagnostic genetic testing to clinical patient groups. The main objective of this quality initiative project is to decrease the cost of NGS send-out tests and to perform patient testing in-house. The CGL NGS workflow and processes must be validated to comply with the clinical accreditation requirements of the College of American Pathologists (CAP). Methods/Case Report Assay performance metrics are considered for the validation of the Illumina NovaSeq 6000 NGS platform. Sample library preparation is performed in PG while sequencing and data analysis is based in CGL. NGS quality assurance in CGL involves the use of Illumina PhiX control mixed with the pooled library. To maintain the high throughput and low-cost NGS approach, Illumina S4 flow cells are used with efficient library pooling techniques. The TAT of clinical samples for WES and WGS analysis in CGL is around 14 days which includes sequencing, data conversion, and bioinformatics. Results (if a Case Study enter NA) A Service Level Agreement (SLA) between Sidra Research and Sidra Pathology was created to define the workflow and services provided by both departments with clear lines of responsibility and accountability. A quality manual is also written to describe the QMS of the genomics laboratory and outlines the various NGS services offered. CGL protocols are transferred to CAP-approved SOP templates for standardization with pathology documentation. For quality improvement, the NGS workflow in CGL is continuously developed to improve the data produced and TAT. Conclusion The collaboration between the two departments maximized the available infrastructures of Sidra Medicine, saved costly send-out tests, improved the TAT of genetic analysis, and most importantly enhanced healthcare outcomes and related clinical diagnostics in alignment with the National Health Strategy (NHS) of the country. Accreditation propels good QMS in laboratory services hence this quality initiative fulfills Sidra Medicine’s vision to provide total precision diagnosis and personalized medicine leading to the delivery of exceptional care to rank amongst the top medical centers internationally.

Involvement of the JAK-STAT Pathway in the Molecular Landscape of Fusion-Free Myeloid Neoplasms with Eosinophilia

Introduction. Gene fusions leading to the constitutive activation of tyrosine kinases (TK) such as PDGFRA, PDGFRB or FGFR1 have been the first recurrent genetic defects involved in myeloid hypereosinophilic syndromes (HES). Although activation of the Janus kinase (JAK)/Signal Transducer and Activator of Transcription(STAT) pathway is critical for eosinophil production and survival, genes involved in the JAK/STAT pathway are not included in most next-generation sequencing (NGS) panels used for the etiological workup of hypereosinophilia (HE). Methods. A custom 149-gene NGS panel including subunits of the IL3/IL5/GM-CSF receptors, TK ( PDGFRA/B, FGFR1, ABL1, FLT3, KIT), intracellular proteins of the JAK-STAT and RAS-MAPK pathways was performed in 64 consecutive adult patients (experimental group) referred between March 2012 to June 2023 to the French Reference Center for Hypereosinophilic Syndromes (CEREO) for HE/HES displaying at least one clinic-biological feature suggestive of myeloid neoplasm ( i.e. splenomegaly, other unexplained CBC abnormality besides HE, increased serum tryptase and/or vitamin B12 levels, corticosteroid-refractory HE and/or sensitivity to either TK inhibitors or JAK inhibitors). All of them were negative by PCR and/or FISH analyses for gene rearrangements involving PDGFRA, PDGFRB or FGFR1. Patients with lymphocytic HES (n=7), idiopathic HES (n=26) and HE of undetermined significance (n=11) were used as controls (total, n=44). Results. Concordant with the latest recommendations of the international cooperative working group on eosinophil-associated disorders, at least one mutation was reported in 50/64 (78%) patients of the experimental group versus 8/44 (18%) patients in the control group (p<0.001) when applying a threshold of at least 3% of variant allele frequency (VAF). Mutations in the control group implied genes involved in age-related clonal hematopoiesis ( e.g. DNMT3A, TET2), with low VAF in almost all cases. All 35 patients with at least one mutation involving the JAK/STAT pathway belonged to the experimental group, among whom all 22 patients treated with steroids were refractory to therapy. Eighteen patients had STAT5B mutations, including 13 (72%) with the somatic N642H mutational hotspot. Two patients harbored JAK2Ex13InDel mutations, including one with eosinophilia and erythrocytosis. Previously unreported molecular alterations were also evidenced, including seven patients with JAK1 mutations and three STAT5A-mutated patients who shared common features i.e. the co-occurrence of BCOR mutations, high hemoglobin levels and eosinophil hyperplasia. Of note, the latter mutations were not reported both in a public database (GnomAD) as well as in a second cohort of 613 samples referred for suspicion of myeloid malignancies (yet without HE) studied with the same NGS workflow, thereby strongly supporting their association with myeloid HES. Deciphering the data from bulk sequencing also suggests that JAK-STAT mutations were frequently preceded by (or associated with) myelodysplasia-related gene mutations, with SF3B1 (12/36) and ASXL1 (10/36) mutations being the most common. Overall, both KIT D816V (n=2) and RAS/MAPK pathway activation mutations (n=3) were rare. No mutation of either PDGFRA/B, FGFR1 or IL3/IL5/GM-CSF-receptor genes was evidenced.In the experimental group, 17/18 (94%) patients (including 12 with JAK-STAT mutations) treated with ruxolitinib and with > 3 months of follow-up responded to treatment (12 complete and 5 partial hematological responses). Discussion. These dataemphasize the usefulness of NGS in daily practice for the workup of fusion-free HES patients harboring features suggestive of myeloid neoplasms. In such patients, druggable mutations involving the JAK-STAT pathway (including yet unidentified STAT5A mutations) are frequent. Most JAK/STAT mutations occur in the setting of a preexisting myelodysplastic or myelodysplastic/myeloproliferative disease harboring mutations in RNA-splicing genes or chromatin modifiers. These findings provide a rationale for refining treatment algorithms in fusion-free myeloid HES patients, supporting the use of JAK inhibitors as frontline therapy. More data are warranted to assess whether JAK inhibition enables sustained molecular remission in all disease subtypes.

Ultra-Fast Amplicon-Based Next-Generation Sequencing in Non-Squamous Non-Small Cell Lung Cancer.

The number of molecular alterations to be tested for targeted therapy of non-squamous non-small cell lung cancer (NS-NSCLC) patients has significantly increased these last few years. The detection of molecular abnormalities is mandatory for the optimal care of advanced or metastatic NS-NSCLC patients, allowing targeted therapies to be administrated with an improvement in overall survival. Nevertheless, these tumors develop mechanisms of resistance that are potentially targetable using novel therapies. Some molecular alterations can also modulate the treatment response. The molecular characterization of NS-NSCLC has to be performed in a short turnaround time (TAT), in less than 10 working days, as recommended by the international guidelines. In addition, the origin of the tissue biopsies for genomic analysis is diverse, and their size is continuously decreasing with the development of less invasive methods and protocols. Consequently, pathologists are being challenged to perform effective molecular technics while maintaining an efficient and rapid diagnosis strategy. Here, we describe the ultra-fast amplicon-based next-generation sequencing (NGS) workflow used in daily routine practice at diagnosis for NS-NSCLC patients. We showed that this system is able to identify the current molecular targets used in precision medicine in thoracic oncology in an appropriate TAT.

SDPS-44 RAPID DETECTION OF MUTATIONS IN CSF-CFTNA WITH THE GENEXUS INTEGRATED SEQUENCER

Abstract Brain metastases commonly arise in patients with lung and breast carcinomas and pose distinctive clinical challenges. Genomic alterations are fundamental for diagnosis and therapy selection in cancer patients. However, the turn-around-time (TAT) of standard next generation sequencing (NGS) assays is a limiting factor in the timely delivery of genomic information for clinical decision making. The GenexusTM Sequencer automates the NGS workflow delivering results in less than 24hrs. The Oncomine Precision Assay (OPA) evaluates somatic mutations, copy number variations and fusions in hot-spot regions of 50 cancer-related genes. In this study, we evaluated genomic alterations in 46 cerebrospinal fluid (CSF) samples from 33 patients with metastatic lung cancer (mLC) (N=32) and metastatic breast cancer (mBC) (N=14) to the brain. Cell-free total nucleic acids, (cfTNA) were extracted using QIAamp Circulating Nucleic Acid kit with cfDNA concentrations ranging from 0.1-11.2(ng/ul) and 0.5-6.6 (ng/ul) for mLC and mBC samples, respectively. Median base coverage was 37,913X and 33,576.5X for mLC and mBC samples, respectively, with cfDNA input ranging from 2-20ng. Mutations were detected in 15/32 (46.8%) mLC and 8/14 (57.1%) mBC CSF samples. Among the samples with no mutations detected, 18/23 (78.2%) had suboptimal DNA input (<20ng). The EGFR exon 19 deletion was detected in 4 samples from one patient, with increasing mutant allele fraction in CSF over time, highlighting the potential of CSF-cfTNA analysis for monitoring patients. Moreover, the EGFR T790M mutation was detected in a patient with prior EGFR inhibitor treatment. In addition, ESR1 D538G and ESR1::CCDC170 alterations, associated with endocrine therapy resistance, were detected in 2 mBC patients. The average TAT from cfTNA-to-results was ~16hrs. In summary, our results indicate that CSF-cfTNA analysis with the OPA in the Genexus instrument can provide clinically relevant information in patients with brain metastases with a short TAT.

Emerging Genomic Trends on Rabies Virus in Davao Region, Philippines, 2018-2021.

Rabies, caused by the rabies virus (RABV), remains a significant public health issue in the Philippines despite efforts to control it. To eliminate rabies by 2030, effective surveillance strategies are crucial. In this study, we examined RABV evolution and phylodynamics in the Davao Region using genome sequences from Davao City and nearby provinces. We adapted the RABV ARTIC Protocol for Oxford Nanopore High-Throughput Sequencing to optimize workflow efficiency under limited resources. Comparing new virus samples collected from June 2019 to June 2021 (n = 38) with baseline samples from June 2018 to May 2019 (n = 49), new sub-clades were observed in the phylogenetic tree, suggesting divergence from older variants that were previously undetected. Most of the new viruses belonged to the Asian SEA4_A1.1.1 lineage, but new (SEA4_B1 and SEA4_B1.1) and emerging (SEA4_B1.1_E1) lineages that have never been reported in the Philippines were also identified. The baseline study reported phylogeographic clustering of RABV isolates from the same areas. However, this pattern was disrupted in the current biosurveillance, with variants detected in areas outside the original cluster. Furthermore, our findings revealed significant transmission routes between Davao City and neighboring provinces, contrasting with the predominantly intra-city transmission observed in the baseline study. These results underscore the need for ongoing and timely genomic surveillance to monitor genetic diversity changes and the emergence of novel strains, as well as to track alterations in transmission pathways. Implementing cost-effective next-generation sequencing workflows will facilitate the integration of genomic surveillance into rabies control programs, particularly in resource-limited settings. Collaborations between different sectors can empower local laboratories and experts in genomic technologies and analysis.

Report of the third conference on next-generation sequencing for adventitious virus detection in biologics for humans and animals

Next-generation sequencing (NGS) has been proven to address some of the limitations of the current testing methods for adventitious virus detection in biologics. The International Alliance for Biological Standardization (IABS), the U.S. Food and Drug Administration (FDA), and the European Directorate for the Quality of Medicines and Healthcare (EDQM) co-organized the “3rd Conference on Next-generation Sequencing for Adventitious Virus Detection in Biologics for Humans and Animals”, which was held on September 27–28, 2022, in Rockville, Maryland, U.S.A. The meeting gathered international representatives from regulatory and public health authorities and other government agencies, industry, contract research organizations, and academia to present the current status of NGS applications and the progress on NGS standardization and validation for detection of viral adventitious agents in biologics, including human and animal vaccines, gene therapies, and biotherapeutics. Current regulatory expectations were discussed for developing a scientific consensus regarding using NGS for detection of adventitious viruses. Although there are ongoing improvements in the NGS workflow, the development of reference materials for facilitating method qualification and validation support the current use of NGS for adventitious virus detection.

Improved recovery of SARS-CoV-2 from wastewater through application of RNA and DNA stabilising agents.

Wastewater Based Epidemiology (WBE) has become an integral part of the public health effort to track the levels of SARS-CoV-2 within communities. Detection of SARS-CoV-2 in wastewater can be challenging due to relatively low levels of virus within the sample. The wastewater matrix is also comprised of commercial and domestically derived contaminants, as well as RNases, all of which can adversely affect RT-qPCR analysis. To improve SARS-CoV-2 detection within wastewater samples we investigated both the effect of template dilution (as a means to reduce RT-qPCR inhibition) and sample stabilisation via addition of DNA/RNA Shield™ and/or RNA Later™ (to prevent RNA degradation via RNases) as a means to improve viral fragment detection. Using both methodologies, a significant improvement in SARS-CoV-2 detection from wastewater samples was observed. No adverse effects of stabilising agent addition on downstream Next-Generation Sequencing workflows were detected.

Towards a Rapid-Turnaround Low-Depth Unbiased Metagenomics Sequencing Workflow on the Illumina Platforms.

Unbiased metagenomic sequencing is conceptually well-suited for first-line diagnosis as all known and unknown infectious entities can be detected, but costs, turnaround time and human background reads in complex biofluids, such as plasma, hinder widespread deployment. Separate preparations of DNA and RNA also increases costs. In this study, we developed a rapid unbiased metagenomics next-generation sequencing (mNGS) workflow with a human background depletion method (HostEL) and a combined DNA/RNA library preparation kit (AmpRE) to address this issue. We enriched and detected bacterial and fungal standards spiked in plasma at physiological levels with low-depth sequencing (<1 million reads) for analytical validation. Clinical validation also showed 93% of plasma samples agreed with the clinical diagnostic test results when the diagnostic qPCR had a Ct < 33. The effect of different sequencing times was evaluated with the 19 h iSeq 100 paired end run, a more clinically palatable simulated iSeq 100 truncated run and the rapid 7 h MiniSeq platform. Our results demonstrate the ability to detect both DNA and RNA pathogens with low-depth sequencing and that iSeq 100 and MiniSeq platforms are compatible with unbiased low-depth metagenomics identification with the HostEL and AmpRE workflow.

A CRISPR-enhanced metagenomic NGS test to improve pandemic preparedness

The lack of preparedness for detecting and responding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen (i.e., COVID-19) has caused enormous harm to public health and the economy. Testing strategies deployed on a population scale at day zero, i.e., the time of the first reported case, would be of significant value. Next-generation sequencing (NGS) has such capabilities; however, it has limited detection sensitivity for low-copy-number pathogens. Here, we leverage the CRISPR-Cas9 system to effectively remove abundant sequences not contributing to pathogen detection and show that NGS detection sensitivity of SARS-CoV-2 approaches that of RT-qPCR. The resulting sequence data can also be used for variant strain typing, co-infection detection, and individual human host response assessment, all in a single molecular and analysis workflow. This NGS work flow is pathogen agnostic and, therefore, has the potential to transform how large-scale pandemic response and focused clinical infectious disease testing are pursued in the future.