Library Preparation Research Articles

BIOM-28. WHOLE EXOME TUMOR MOLECULAR PROFILING FROM CEREBROSPINAL FLUID (CSF): PRELIMINARY RESULTS OF A PILOT STUDY

Abstract BACKGROUND Tissue sampling for molecular profiling has become an integral part of the diagnosis, prognosis, and management of CNS malignancies. However, many tumors are surgically inaccessible, and even biopsy of accessible lesions conveys some risk, and the possibility of sampling error. We evaluated the accuracy and potential utility of CSF liquid biopsy for identifying mutations in patients with intracranial tumors. METHODS CSF samples were collected from ventricular reservoirs and stored at -80 degrees Celsius until analyzed. Cell-free nucleic acids were extracted, concentrated, and quantified. Samples over 110ng quantification were normalized to 110ng and input into library prep. Library prep was done using v3.5 of Caris Assure and whole exome sequencing in an S2 FC on a Novaseq 6000. Bioinformatic analysis was performed. RESULTS CSF samples were obtained from 20 patients (10 gliomas, 5 CNS lymphomas, 5 metastases), 19 of whom also had tumor specimens available. Two hundred eighty-one mutations were identified: 17 (6.6%) in tumor only, 184 (72.4%) in CSF only, and 80 (31.5%) in both. Key alterations found in CSF include: IDH, TERT, ATRX in gliomas, and MYD88, PIM1, BCL2, TERT in lymphomas. Glioma CSF with IDH1 mutations did not have PTEN mutations (with one exception, in which the CSF was collected 3 years after the tissue). Targetable mutations in metastases included PIK3CA, NF1, and BRCA. CONCLUSION These preliminary results suggest that CSF liquid biopsy may be useful for identifying mutations in primary and metastatic brain tumors. Serial sampling is feasible, and may disclose changes in diagnostic and/or driver mutations over time, with important therapeutic and diagnostic implications. Many mutations were found in CSF alone. CSF may capture the entire molecular landscape of the tumor, overcoming the sampling error inherent in tissue biopsies. Prospective studies are underway to further elucidate the clinical applications of this technique.

BIOM-04. SINGLE-CELL RNA SEQUENCING PROTOCOL OF THE HUMAN METASTATIC PROSTATE SPINE TUMOR MICROENVIRONMENT

Abstract INTRODUCTION 90% of spine tumor patients can develop metastases with debilitating complications, such as sensorimotor dysfunction, paralysis, and spinal instability. As spine tumor patients have limited eligibility to participate in clinical trials and have heterogenous tumor pathology, there is a distinct lack of therapeutic targets and prognostication markers of disease. Thus, there is a distinct need for molecular profiling of spine tumors. OBJECTIVE We describe an OR-to-bench-top processing of spine tumors for single-cell omic studies, with samples from a metastatic prostate cancer patient to demonstrate the ability to compare molecular markers and microenvironments of difficult-to-process spine tumors and adjacent normal tissue. METHODS Under IRB protocol #Pro00101198, patients undergoing spine surgery with the Department of Neurosurgery at Duke University were consented for tissue collection. Vertebral osseous metastases and adjacent normal tissue were confirmed histologically (H&E, immunohistochemistry) and radiologically (MRI, PET, CT, and x-ray). Tissues were manually and enzymatically homogenized in serum-free media with collagenase A and DNAse I to obtain a single cell suspension. Cells were then filter sterilized, erythrolyzed, counted to determine live-death ratio, resuspended to 10-20 million cells per mL, then cryopreserved for downstream standard library preparation for single-cell sequencing using the 10X genomics platform. Downstream analysis was performed using Seurat pipeline, InferCNV analysis, and SingleR. RESULTS P404S and PSA expression confirmed tumor was prostate in origin. Radiological imaging and H&E staining confirmed the presence of T7 osseous metastasis. Uniform manifold approximation and project (UMAP) plots identified 12 unique clusters of immune cell subpopulations between normal and tumorous vertebrae, with their own subsets of differentially expressed genes. CONCLUSION We have developed a protocol to generate granular single cell molecular profiling for spine tumors and metastases. Applications of this protocol can aid in identifying therapeutic targets, molecular predictors of disease, and potentiate increased eligibility of patients for clinical trials.

Rapid diagnosis of Mycoplasma pneumoniae and prediction of antibiotic resistance by nanopore adaptive sampling

AbstractBackgroundMicrobial infections, particularly in children, require rapid and accurate diagnostics. It is difficult to differentiate pathogens from commensal organisms, and it is impossible to identify antibiotic resistance genes that belong to pathogens with current methods. Third‐generation sequencing provides rapid library preparation and real‐time data acquisition. Nanopore normal sampling (NNS) enables unbiased sequencing of clinical samples without amplification, aiding pathogen identification and antimicrobial resistance gene prediction. However, clinical samples often contain a considerable amount of human DNA, potentially masking pathogen data. Nanopore adaptive sampling (NAS) aims to selectively enrich pathogens, promising improved diagnostics for acute infections and better treatment decisions in clinical practice. This study aimed to determine the utility of NAS in enhancing the real‐time detection of pathogens and predicting AMR in infectious disease outbreaks.MethodsThis study used NAS technology to rapidly and directly detect Mycoplasma pneumoniae infection in bronchoalveolar lavage fluid samples from 28 pediatric patients at Shenzhen Children's Hospital. We assessed the efficacy of NAS compared with that of NNS by evaluating the number of microbial reads and the amount of microbial DNA data. We then compared the accuracy of detecting pathogens between NNS and NAS and between NAS and real‐time polymerase chain reaction assays. Furthermore, we predicted antimicrobial resistance (AMR) and examined AMR genes associated with pathogens.ResultsNAS showed up to a 14.67‐fold increase in the amount of microbial DNA data from patients' samples compared with NNS within the initial 2.5 h of sequencing. Additionally, NAS reduced the amount of host DNA data by up to 6.67‐fold compared with NNS. Unlike TaqMan real‐time polymerase chain reaction assays, NAS technology identified dominant pathogens and provided detailed insight into the abundance of the microbial community. Furthermore, NAS was able to predict AMR profiles of microbial communities and attribute specific AMR traits to individual microbes within the samples.ConclusionThis study shows that NAS advances the clinical diagnosis because it can rapidly detect pathogens directly from patients' samples and provides antimicrobial resistance information for clinical guidance. These abilities further facilitate the application of NAS in personalized treatment, reduce the misuse of broad‐spectrum antibiotics, and promote patients' recovery.

In Vitro Genome-Wide Identification of Transcription Factor Binding Sites.

DNA affinity purification followed by sequencing (DAP-seq) profiles transcription factor (TF) binding sites in vitro, in which recombinant TF captures the genomic fragments with TF binding sites. Here, I describe the preparation of pre-PCR DNA-seq libraries for 96 TFs, including the expression and purification of recombinant TF, the affinity purification of TF binding fragments, and the sequencing-ready library preparation. Using this method, up to 96 DAP-seq libraries can be prepared in 2days.

Whole exome sequencing: the search for mutations associated with hereditary breast cancer in ethnic groups of Siberia

Hereditary breast cancer (HBC) is a heterogeneous disease caused by mutations in genes characterized by ethnic specifcity. The clinical heterogeneity of this disease signifcantly complicates its diagnosis. The use of high-throughput sequencing is one of the approaches that allow the search for genes and their variants associated with the development of HBC. The purpose of the study was to search for new genes associated with HBC in the understudied ethnic groups of Siberia by using whole exome sequencing (WES).Material and Methods. WES was performed on a cohort of 16 probands with BC (Tuvan, Yakut, Altai ethnos). The study material was genomic DNA isolated from peripheral blood leukocytes. Libraries were prepared using a BGI Optimal DNA Library Prep kit. An Agilent SureSelect Human All Exon V6 kit was used for hybridization. High-throughput sequencing was performed on a DNA nanoball sequencing platform (DNBSeq-G400).Results. In the overall group of patients with signs of HBC, pathogenic variants were detected in 12.5 % of cases (2/16). For the frst time, BRCA1 (rs80357635) pathogenic variant was identified in a young patient with metachronous BC (Yakut ethnic group). A pathogenic variant of the ATM gene (rs780619951 NM_000051:exon16:c.C2413T:p.R805X) was identified in a young patient with BC (Tuvinian ethnic group). A pathogenic variant of the TDP2 c.G4T:p.E2X, rs770844602 gene (DNA repair gene) was identified for the frst time in a Tuvan BC patient (metachronous) with a family history, but its contribution to HBC remains to be proven. The TDG gene variant (rs764159587 NM_001363612:exon7:c.536dupA:p.E179fs) found in the Tuvan ethnic group and affecting splicing (SpliceAI: 0.580) requires special attention.Conclusion. This report is the frst to describe the germinal variant in the BRCA1 (rs80357635) gene in the Yakut ethnic group. Further studies are required to confrm pathogenicity of germinal variants in non-well studied genes TDP2, TDG in ethnic BC patients.

DeLTa-Seq: High-Throughput Targeted RNA-Seq of Rice Leaves Without RNA Purification.

DeLTa-seq is a high-throughput RNA-seq library preparation method that enables quantification of the expression of hundreds of arbitrarily selected genes without RNA purification. This method involves direct reverse transcription using rice leaf lysate and targeted RNA-seq library preparation. DeLTa-seq enables the precise quantification of gene expression with a small number of sequencing reads. This chapter provides detailed information on the design of gene-specific primers, sampling of rice leaves, preparation of lysates, direct-lysate reverse transcription, targeted RNA-seq library preparation, and bioinformatic analysis of DeLTa-seq data.

Cross-comparison of gut metagenomic profiling strategies

The rapid advancements in sequencing technologies and bioinformatics have enabled metagenomic research of complex microbial systems, but reliable results depend on consistent laboratory and bioinformatics approaches. Current efforts to identify best practices often focus on optimizing specific steps, making it challenging to understand the influence of each stage on microbial population analysis and compare data across studies. This study evaluated DNA extraction, library construction methodologies, sequencing platforms, and computational approaches using a dog stool sample, two synthetic microbial community mixtures, and various sequencing data sources. Our work, the most comprehensive evaluation of metagenomic methods to date. We developed a software tool, termed minitax, which provides consistent results across the range of platforms and methodologies. Our findings showed that the Zymo Research Quick-DNA HMW MagBead Kit, Illumina DNA Prep library preparation method, and the minitax bioinformatics tool were the most effective for high-quality microbial diversity analysis. However, the effectiveness of pipelines or method combinations is sample-specific, making it difficult to identify a universally optimal approach. Therefore, employing multiple approaches is crucial for obtaining reliable outcomes in microbial systems.

Lasy-Seq: A High-Throughput 3' RNA-Seq Method for Large-Scale Transcriptome Profiling in Rice.

Lasy-Seq (low-cost and easy RNA-seq) is a high-throughput library preparation method for 3' RNA-seq. Usage of oligo(dT) primers with unique index sequences allows simultaneous reverse transcription (RT) reaction and sample indexing. Therefore, each post-RT step, which was performed on a sample-by-sample basis in a conventional method, can be performed on a large number of samples together in a single tube. With the Lasy-Seq, the sequencing-ready library pool can be prepared from a few to several hundred samples in a few days.

Profiling of 24-nt phasiRNAs in Rice Anther by Massively Parallel Sequencing and Stem-Loop Reverse Transcription PCR.

Small RNAs are highly abundant and play important roles in plant reproduction. Profiling of small RNAs in reproductive tissues is a critical step in understanding their biology. Here, we describe a protocol for small RNA profiling in rice anthers, with a focus on an abundantly expressed but little-understood reproductive small RNA class named 24-nucleotide phased secondary small interfering RNAs (24-nt phasiRNAs). The protocol details small RNA library preparation steps for high-throughput sequencing, with a subsequent bioinformatic analysis framework. A low-throughput stem-loop reverse transcription PCR protocol for quick semi-quantification of small RNAs is also included as a complementary approach. The collective protocol, although focused on 24-nt phasiRNAs, should be amenable to other small RNA classes in various plant reproductive tissues.

Genomic Proximity Mapping (GPM): Evaluation of a Next Generation Cytogenomic Assay for Improved Risk Stratification in Acute Myeloid Leukemia

Cytogenetics encompasses a suite of diagnostics assays that provides critical diagnostic and prognostic information that can help guide care for acute myeloid leukemias (AML) patients. Routine cytogenetic workup for AML includes karyotyping and fluorescence in situ hybridization (FISH) for known recurrent variants. The workup can be extended to include chromosome genome array testing (CGAT) but because all 3 of these tests have a non-overlapping set of limitations that can necessitate multiple, often iterative, rounds of testing in complex cases and may miss information in cases with cryptic fusions or small copy number alterations. Therefore, there is an unmet need for an accurate, fast and reliable test that can provide a solution to these limitations. In this study, we evaluate Genomic Proximity Mapping (GPM), a novel method that captures much of the same information as karyotyping, FISH, and CGAT in a single assay. GPM uses proximity ligation sequencing to capture ultra-long range contiguity information from conventional short-read sequencing. In brief, 200,000-500,000 cells from bone marrow, peripheral blood, or aphersis samples are crosslinked with formalin prior to library preparation. Because sequences that are closer on a chromosome are more likely to physically interact and be crosslinked, GPM can use the frequency pairwise of crosslinked sequence interactions to determine the structure of chromosomes. In order to evaluate its performance, we benchmarked GPM against standard-of-care cytogenetics conducted on newly diagnosed 166 AML cases in a multicenter, retrospective study utilizing real-world patient samples. There was a strong concordance between GPM and cytogenetic findings, with 100% of European LeukemiaNet (ELN) specified cytogenetic risk variants identified by cytogenetic being identified by GPM. However, GPM also identified additional variants of known clinical significance not observed by standard-of-care cytogenetics. To determine if GPM findings had clinical benefits for risk stratification, we compared overall survival (OS) of patients, risk-stratified using ELN 2022 criteria, using either cytogenetics or GPM to determine the cytogenetic abnormalities. Separation of favorable, intermediate, and adverse risk categories were statistically significant when variants identified either by cytogenetics or GPM were used for risk categorization (GPM, P = 0.0006; cytogenetics P = 0.0009 for GPM and cytogenetics, Mantel-Cox log rank test). However, Kaplan-Meier analysis demonstrated that GPM greater separation in intermediate (median OS 2.1 years for GPM and 2.4 years for cyto), and adverse (median OS 1.1 yrs for GPM and 0.9 years for cytogenetics) categories. The ELN-directed risk based on karyotype and FISH presentation was revised for 14 (8.4%) out of 166 cases with available data based on GPM assay results. These findings argue that GPM can be used as a single assay to accurately stratify AML patient risk and may provide additional benefits over cytogenetics, yielding more accurate risk stratification. Support: NIH/NCI U10CA180888, U10CA180819, U24CA196175, R44CA278140

Single Laboratory Evaluation of the Q20+ Nanopore Sequencing Kit for Bacterial Outbreak Investigations.

Leafy greens are a significant source of produce-related Shiga toxin-producing Escherichia coli (STEC) outbreaks in the United States, with agricultural water often implicated as a potential source. Current FDA outbreak detection protocols are time-consuming and rely on sequencing methods performed in costly equipment. This study evaluated the potential of Oxford Nanopore Technologies (ONT) with Q20+ chemistry as a cost-effective, rapid, and accurate method for identifying and clustering foodborne pathogens. The study focuses on assessing whether ONT Q20+ technology could facilitate near real-time pathogen identification, including SNP differences, serotypes, and antimicrobial resistance genes. This pilot study evaluated different combinations of two DNA extraction methods (Maxwell RSC Cultured Cell DNA kit and Monarch high molecular weight extraction kits) and two ONT library preparation protocols (ligation and the rapid barcoding sequencing kit) using five well-characterized strains representing diverse foodborne pathogens. High-quality, closed bacterial genomes were obtained from all combinations of extraction and sequencing kits. However, variations in assembly length and genome completeness were observed, indicating the need for further optimization. In silico analyses demonstrated that Q20+ nanopore sequencing chemistry accurately identified species, genotype, and virulence factors, with comparable results to Illumina sequencing. Phylogenomic clustering showed that ONT assemblies clustered with reference genomes, though some indels and SNP differences were observed, likely due to sequencing and analysis methodologies rather than inherent genetic variation. Additionally, the study evaluated the impact of a change in the sampling rates from 4 kHz (260 bases pair second) to 5 kHz (400 bases pair second), finding no significant difference in sequencing accuracy. This evaluation workflow offers a framework for evaluating novel technologies for use in surveillance and foodborne outbreak investigations. Overall, the evaluation demonstrated the potential of ONT Q20+ nanopore sequencing chemistry to assist in identifying the correct strain during outbreak investigations. However, further research, validation studies, and optimization efforts are needed to address the observed limitations and fully realize the technology's potential for improving public health outcomes and enabling more efficient responses to foodborne disease threats.

Genomic Profiling of Two Successive Post-Transplant Lymphoproliferative Disorders with Different Morphology in a Pediatric Lung Transplant Recipient

Introduction Post-transplant lymphoproliferative disorders (PTLD) encompass a broad spectrum of lymphoid proliferations, a consequence of externally induced immunosuppression following solid organ or hematopoietic stem cell transplantation. These disorders range from indolent proliferations to polyclonal polymorphic diseases, but they can also present as aggressive malignant lymphomas that highly resemble those observed in the immunocompetent population. Due to the shared pathogenetic mechanisms, studying PTLDs can provide valuable insights into the pathogenesis of lymphomas in general. One of the most intriguing real-life models of lymphomagenesis represent patients who develop multiple PTLDs with different morphologies, even though such cases are extremely rare. Aims In this study, we focused on a case of a lung transplant patient who gradually developed two PTLDs with different morphologies. Our aim was to evaluate the relationship between these two diseases and to elucidate the morphological progression. Methods A female EBV seronegative patient with cystic fibrosis underwent a lung transplantation from EBV seropositive donor at 16 years of age. She first developed polymorphic PTLD in the colon 11 months post-transplantation (CD20+CD30-EBV+). She was treated with reduction of immunosuppression and 6 doses of rituximab according to Ped-PTLD-2005-Pilot protocol. The clinical status of the patient improved significantly and a PET-CT scan performed after the 4th dose of rituximab showed marked regression of all the lesions. However, 15 months post-transplantation, she presented with diffuse large B-cell lymphoma (DLBCL) in the duodenum (CD20+CD30+EBER+). The patient was treated with R2 arm of NHL-BFM 2004 protocol and has achieved long-lasting remission of the disease. DNA was isolated from formalin-fixed, paraffin-embedded (FFPE) samples of polymorphic PTLD and DLBCL using QIAamp DNA FFPE Tissue kit (Qiagen). Immunoglobulin (IG) rearrangements were analyzed by Biomed-2 IGH FR3 Multiplex PCR. Allele-specific oligonucleotide quantitative PCR was designed based on monoclonal IGH sequence from DLBCL to backtrack the clone in PTLD and in peripheral blood (PB) samples with sensitivity of 1E^-04. Whole exome sequencing (WES) was performed on DNA from FFPE PTLD biopsy samples and pre-transplant PB using the SureSelect XT HS2 Human All Exon V8+UTRs library preparation kit (Agilent Technologies) and NextSeq 500 instrument (Illumina). Bioinformatic analysis was performed using Varsome Clinical. Variants not present in germline with sufficient coverage and sequencing quality predicted as pathogenic or of uncertain significance were filtered out. Results An identical monoclonal IGHV4-39-IGHJ4 rearrangement was detected in biopsy samples of both PTLDs. This rearrangement was also detected in PB two weeks prior to the DLBCL diagnosis. PB samples (n=10) taken at previous time points before and after transplantation were all negative. WES identified 103 somatic variants in total: 32 were present only in polymorphic PTLD, 60 only in DLBCL, and 11 were shared between the two diseases. Of the 26 variants with the highest predicted pathogenicity, only 5 were present in polymorphic PTLD and 25 in DLBCL. Notably, variants in the FAS, KMT2C, and LRP1B genes were detected in DLBCL but not in polymorphic PTLD. Variants in these genes have been previously described in pediatric PTLD-DLBCL (Salmerón-Villalobos et al., Blood, 2024) and in adult refractory DLBCL and transformed follicular lymphoma (Morin et al., Clinical Cancer Research, 2016), often occurring concurrently, suggesting synergistic effect. Conclusions The polymorphic PTLD and DLBCL developed from the same B-cell clone with an identical IGH rearrangement. The progression to DLBCL was most likely driven by the selection of a clone with newly acquired somatic variants in the FAS, KMT2C, and LRP1B genes. To our knowledge, only a few cases of multiple PTLDs with different morphologies in a single patient have been described in the literature, and none have been evaluated to this extent at the genomic level. This case highlights that PTLDs can provide valuable insights into the pathogenesis of NHLs in general. Supported by the European Union - Next Generation EU - program No. LX22NPO5102, Charles University Research Centre program No. UNCE/24/MED/003 and MH CZ - DRO, Motol University Hospital, Prague, Czech Republic 00064203

CIRCLE-Seq for Interrogation of Off-Target Gene Editing.

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of unintended cleavage sites of CRISPR-Cas9 through targeted sequencing of CRISPR-Cas9 cleaved DNA. The protocol involves circularizing genomic DNA (gDNA), which is subsequently treated with the Cas9 protein and a guide RNA (gRNA) of interest. Following treatment, the cleaved DNA is purified and prepared as a library for Illumina sequencing. The sequencing process generates paired-end reads, offering comprehensive data on each cleavage site. CIRCLE-seq provides several advantages over other in vitro methods, including minimal sequencing depth requirements, low background, and high enrichment for Cas9-cleaved gDNA. These advantages enhance sensitivity in identifying both intended and unintended cleavage events. This study provides a comprehensive, step-by-step procedure for examining the off-target activity of CRISPR-Cas9 using CIRCLE-seq. As an example, this protocol is validated by mapping genome-wide unintended cleavage sites of CRISPR-Cas9 during the modification of the AAVS1 locus. The entire CIRCLE-seq process can be completed in two weeks, allowing sufficient time for cell growth, DNA purification, library preparation, and Illumina sequencing. The input of sequencing data into the CIRCLE-seq pipeline facilitates streamlined interpretation and analysis of cleavage sites.

Development of a Tagmentation-Based Next-Generation Sequencing Clinical Assay as an Alternative to Capillary Electrophoresis-Based Sequencing.

Next-generation sequencing (NGS) technology enables sample multiplexing for interrogation of multiple regions of interest (ROI). Leveraging this, together with access to affordable NGS platforms, we explored the practicality of moving capillary electrophoresis (CE), noncapillary electrophoresis and single-gene testing to NGS. In this work, we evaluated the iSeq 100's capacity to validate 89 samples at once. Genomic DNA was extracted from 89 archival samples of varying specimen types. Polymerase chain reaction (PCR) was done with in house primers, library preparation with the Nextera XT Library Preparation Kit and cleaning up with paramagnetic beads. The sequencing was performed on one Illumina iSeq 100 flow cell. With our workflow, 88 out of 89 samples were accurately sequenced with variant alleles identified. One sample of the 88 samples was initially discordant because the primers used were in a heterozygous deletion region. Upon redesigning of primers, the sample proved concordant. The iSeq-Nextera workflow proved accurate. However, variant allele frequencys generated by the Nextera are not precise.

Application of the Agilent 2100 Bioanalyzer instrument as quality control for next-generation sequencing.

Next-generation sequencing (NGS) technologies have expanded the spectrum of forensic DNA analysis by facilitating efficient and precise genotyping of a large number of genetic markers. Yet, challenges persist regarding complex sample processing and assurance of equal molar concentrations across pooled samples. Since optimal cluster density is crucial for sequencing performance, the determination of both quantity and quality is indispensable for library preparation. In this study, we investigated the application of the Agilent 2100 Bioanalyzer for library quality control, as studies for forensic approaches, particularly for highly degraded postmortem samples, are rare. Our analysis encompassed assessing total DNA concentrations, fluorescence unit (FU) values, and adapter dimer concentrations in purified DNA libraries derived from buccal swabs and tissue samples of decomposed corpses. The sensitivity study tested a serial dilution derived from buccal swabs and revealed a decrease in FU values and an increase in adapter dimers with declining DNA input concentrations. Deviations in total DNA concentrations and average peak heights between the Agilent 2100 Bioanalyzer runs indicated a lack of repeatability in data and presented challenges in accurate quantification, which was also observed in previous studies. Yet, the analysis of degraded samples from decomposed human remains has shown the ability to detect adapter dimer concentrations, which can be crucial for the quality of subsequent NGS library preparation and sequencing success. Therefore, the Agilent 2100 Bioanalyzer proves to be a valuable tool for NGS quality control.

Rapid sequencing and identification for 18-STRs long amplicon panel using portable devices and nanopore sequencer

STRs are the most commonly used forensic genetic markers for human identification. Nanopore sequencing has shown the advantages of high portability and large data throughput. Previous studies indicate it has great potential for profiling STRs based on the ligation library preparation method. However, this method, which requires more library preparation time and operations, is unsuitable for rapid STR profiling, particularly for field forensic applications. The transposase-based rapid library preparation method offers the possibility to perform human identification using portable instruments. However, the amplicons of conventional STR panels are too small and would be cut into scraps with rapid methods, making them impractical for genotyping. In this study, we developed an 18-STRs multiplex amplification panel with amplicons of ~1.4 Kbp. The PCR conditions were optimized to be finished within 2 h and 12 min, and the PCR products could undergo rapid methods that involved random fragmentation. We found that, on average, 29.16 % of reads from the long-amplicon panel and rapid library kit covered the whole STR region, sufficient for downstream STR profiling analysis. We conducted a small validation experiment on 24 samples using portable instruments powered by a 1.5 kW‧h portable power source. The entire process took 10.5 h and we obtained enough data from 24 samples to perform trustworthy pairwise identification analysis using the STR profiles. The overall accuracy of the analysis was 95.36 %. In sum, the study evaluated and demonstrated the viability and potential of nanopore sequencing for forensic application in the field.

Distribution and Antimicrobial Resistance of Complicated Intraabdominal Infection Pathogens in Two Tertiary Hospitals in Egypt.

Background: Management of complicated intraabdominal infections (cIAIs) requires containment of the source and appropriate initial antimicrobial therapy. Identifying the local data is important to guide the empirical selection of antimicrobial therapy. In this study, we aimed to describe the pathogen distribution and antimicrobial resistance of cIAI. Methods: In two major tertiary care hospitals in Egypt, we enrolled patients who met the case definition of cIAI from October 2022 to September 2023. Blood cultures were performed using the BACTAlert system (BioMerieux, Marcy l'Etoile, France). A culture of aspirated fluid, resected material, or debridement of the infection site was performed. Identification of pathogens and antimicrobial susceptibility testing were conducted by the VITEK-2 system (BioMerieux, Marcy l'Etoile, France). Gram-negative resistance genes were identified by PCR and confirmed by whole bacterial genome sequencing using the Nextera XT DNA Library Preparation Kit and sequencing with the MiSeq Reagent Kit 600 v3 (Illumina, USA) on the Illumina MiSeq. Results: We enrolled 423 patients, 275 (65.01%) males. The median age was 61.35 (range 25-72 years). We studied 452 recovered bacterial isolates. Gram-negative bacteria were the vast majority, dominated by E. coli, followed by Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Proteus mirabilis (33.6%, 30.5%, 13.7%, 13%, and 5.4%, respectively). High rates of resistance were detected to third- and fourth-generation cephalosporins and fluoroquinolones. No resistance was detected to colistin. Resistance to amikacin and tigecycline was low among all isolates. Resistance to meropenem and ceftazidime/avibactam was moderate. ESBL genes were common in E. coli and K. pneumoniae. CTX-M15 gene was the most frequent. Among Enterobacterales, blaOXA-48 and blaNDM were the most prevalent carbapenemase genes. Pseudomonas aeruginosa isolates harbored a wide variety of carbapenemase genes (OXA, NDM, VIM, SIM, GIM, SPM, IMP, AIM), dominated by metallo-beta-lactamases. In 20.6% of isolates, we identified two or more resistance genes. Conclusion: High resistance rates were detected to third- and fourth-generation cephalosporins and fluoroquinolones. Amikacin and tigecyclines were the most active antimicrobials. Our data call for urgent implementation of antimicrobial stewardship programs and reinforcement of infection control.

Deep Diving Into the Fusion Across Cancer Types in the Indian Population From Formalin-Fixed Paraffin-Embedded RNA-Exome Data: A Road to Discovering Novel Rearrangements With Clinical Relevance.

Gene fusions are critical oncogenic mutations that drive cancer development and serve as diagnostic and prognostic biomarkers. Despite the increasing cancer burden in India, large-scale analyses of molecular landscapes, particularly gene fusions, have been relatively scarce. This retrospective study used RNA-exome data from 1,392 Indian patients with cancer across 15 major cancer types to explore gene fusions. The study used a comprehensive framework that integrated open-source and proprietary tools to detect gene fusions from formalin-fixed paraffin-embedded tumor samples. The process involved RNA extraction, RNA-exome library preparation, and analysis using tools such as FastQC, DRAGEN RNA Pipeline, STAR-Fusion, and FusionInspector. We validated and filtered potential false-positive fusion calls using AGFusion and FusionAnnotator to annotate fusion breakpoints and their functional impact through various in silico tools. The study found a notable prevalence of FGFR fusions across cancer types, especially FGFR3, with FGFR3::TACC3 as the most recurrent. Kinase fusions were prevalent in the cohort accounting for 37% of incidence in the patients. We also identified 91 novel potential driver fusions, including those involving FGFR2, MET, ESR1, and PDGFRA. This study underscores the critical role of gene fusions as biomarkers in cancer, extending beyond fusion-driven malignancies to encompass all cancer types. Gene fusions serve as both diagnostic markers and tumor-agnostic therapeutic targets within the current cancer treatment paradigm. Our insights into the prevalence of oncogenic drivers and novel targets expand the understanding of gene fusions, shedding new light on their mechanisms and clinical implications.

Comparative Analysis of Ficoll-Hypaque and CytoLyt® Techniques for Blood Removal in Breast Cancer Malignant Effusions: Effects on RNA Quality and Sequencing Outcomes

IntroductionTo optimize RNA sequencing (RNA-seq) outcomes, we investigated pre-analytical variables in malignant effusions containing metastatic breast cancer. We compared two processing methods—Ficoll-Hypaque density gradient enrichment and CytoLyt® hemolysis—focusing on their effects on RNA quality, transcript abundance, and variant detection from cytospin slides, relative to fresh-frozen samples. Additionally, we compared read-based and Unique Molecular Identifier-based (UMI) library preparation methods. Materials and MethodsThirteen malignant effusion specimens from metastatic breast cancer were processed using both the Ficoll-Hypaque and Cytolyt® methods. RNA was extracted from fresh-frozen samples stored in RNA preservative and from cytospin slides fixed in Carnoy's solution. RNA quality was evaluated using RNA integrity number (RIN) and the percentage of fragments >200 bases (DV200). Sequencing was conducted with both read and UMI-based methods. ResultsPurified RNA was more fragmented by the Cytolyt® method (mean RIN: 3.56, DV200: 78.97%), compared to the Ficoll-Hypaque method (mean RIN: 6.29, DV200: 88.08%). Sequencing data had high concordance correlation coefficient (CCC) for measurements of gene expression, whether from Cytolyt® or Ficoll-Hypaque treated samples, and whether using the UMI-based or read-based sequencing methods (read-based mean CCC: 0.967 from Cytolyt® versus 0.974 from Ficoll-Hypaque, UMI-based mean CCC: 0.972 from Cytolyt® versus 0.977 from Ficoll-Hypaque). ConclusionsDespite the increased RNA fragmentation with the Cytolyt®, RNA-seq data quality was comparable across Cytolyt® and Ficoll-Hypaque methods. Both clearing methods are viable for short-read RNA-seq analysis, with read and UMI-based approaches performing similarly.

An Oxford Nanopore Technology-Based Hepatitis B Virus Sequencing Protocol Suitable for Genomic Surveillance Within Clinical Diagnostic Settings.

Chronic Hepatitis B Virus (HBV) infection remains a significant public health concern, particularly in Africa, where the burden is substantial. HBV is an enveloped virus, classified into ten phylogenetically distinct genotypes (A-J). Tests to determine HBV genotypes are based on full-genome sequencing or reverse hybridization. In practice, both approaches have limitations. Whereas diagnostic sequencing, generally using the Sanger approach, tends to focus only on the S-gene and yields little or no information on intra-patient HBV genetic diversity, reverse hybridization detects only known genotype-specific mutations. To resolve these limitations, we developed an Oxford Nanopore Technology (ONT)-based HBV diagnostic sequencing protocol suitable for clinical virology that yields both complete genome sequences and extensive intra-patient HBV diversity data. Specifically, the protocol involves tiling-based PCR amplification of HBV sequences, library preparation using the ONT Rapid Barcoding Kit (Oxford nanopore Technologies, Oxford, OX4 4DQ, UK), ONT GridION sequencing, genotyping using genome detective software v1.132/1.133, a recombination analysis using jpHMM (26 October 2011 version) and RDP5.61 software, and drug resistance profiling using Geno2pheno v2.0 software. We prove the utility of our protocol by efficiently generating and characterizing high-quality near full-length HBV genomes from 148 residual diagnostic samples from HBV-infected patients in the Western Cape province of South Africa, providing valuable insights into the genetic diversity and epidemiology of HBV in this region of the world.