Achieving a stable deep molecular response with the option to discontinue tyrosine kinase inhibitor treatment is the new therapeutic goal for patients with chronic myeloid leukemia (CML). Several studies have shown that...Achieving a stable deep molecular response with the option to discontinue tyrosine kinase inhibitor treatment is the new therapeutic goal for patients with chronic myeloid leukemia (CML). Several studies have shown that individuals expressing the BCR::ABL1 e14a2 transcript achieve a major molecular response more rapidly than those with the e13a2 transcript. However, technical issues may have confounded these observations, and data for pediatric patients are limited. This study analyzed the distribution of BCR::ABL1 transcript types and their association with baseline hematologic parameters and tyrosine kinase inhibitor treatment response in 102 pediatric patients with CML. Subgroups were compared on the basis of results from routine multiplex PCR and droplet digital PCR (ddPCR). The dynamics of the transcript types under therapy were evaluated in detail in patients and a CML cell line co-expressing e13a2 + e14a2. ddPCR has identified significantly more patients co-expressing e13a2 + e14a2 than classified on the basis of routine diagnostics. This has implications for the categorization of individual subgroups. Comparing transcript dynamics in individuals or a cell line expressing both variants simultaneously revealed no differences in treatment response. When analyzing clinical data based on the transcript classification of patients, it is important to use methods that detect both variants with equal sensitivity. In ddPCR, the transcript variants' ratio is accurately shown because there is no competitive template amplification, as seen in multiplex and quantitative real-time PCR.
Schumacher S, Malchau Lauesgaard J, Carlsson T
… +2 more, Linder A, Sundfeldt K
J Mol Diagn
· 2025 Mar · PMID 39828035
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Cell-free DNA (cfDNA) of ovarian carcinoma origin can be detected in samples from the gynecologic tract. This study aims to evaluate how pre-analytical handling affects DNA profile and integrity in Papanicolaou (Pap) tes...Cell-free DNA (cfDNA) of ovarian carcinoma origin can be detected in samples from the gynecologic tract. This study aims to evaluate how pre-analytical handling affects DNA profile and integrity in Papanicolaou (Pap) tests, to optimize their potential for detection of ovarian cancers (OCs). Analysis of archived Pap tests from patients with OC, kept at room temperature for 48 hours and stored at -80°C, was complemented by in vitro experiments. Temperature-associated effects on DNA fragmentation were evaluated in samples stored at 4°C, -20°C, or -80°C. Time-dependent DNA degradation at room temperature was evaluated in comparison to storage at 4°C. Results were validated in prospectively collected Pap tests. The DNA integrity was assessed by fragment analysis. Accumulation of short DNA fragments was observed in archived Pap tests from patients with OC. In vitro, fragments of 100 to 350 bp increased 11.5-fold within 48 hours at room temperature compared with 1.7-fold when stored at 4°C. Consistent with the in vitro findings, prospectively collected samples showed reduced fragmentation when stored at 4°C compared with room temperature (P = 0.007). Long-term storage at 4°C had a significant negative effect on DNA stability (P = 0.013), whereas freezing slowed down fragmentation. Immediate storage at 4°C after sampling markedly reduces DNA degradation, suggesting a simple way to optimize pre-analytical handling and decrease unwanted fragmentation for cfDNA analysis in Pap tests.
J Mol Diagn
· 2025 Mar · PMID 39818318
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Single-nucleotide variants (SNVs) and polymorphisms are characteristic biomarkers in various biological contexts, including pathogen drug resistances and human diseases. Tools that lower the implementation barrier of mol...Single-nucleotide variants (SNVs) and polymorphisms are characteristic biomarkers in various biological contexts, including pathogen drug resistances and human diseases. Tools that lower the implementation barrier of molecular SNV detection methods would provide greater leverage of the expanding single-nucleotide polymorphism/SNV database. The oligonucleotide ligation assay (OLA) is a highly specific means for detection of known SNVs and is especially powerful when coupled with PCR. Yet, the OLA design process remains intensive, and criteria for success are uncertain. To assist in the design process, this study describes OLAgen, an open-source tool to automate development of OLAs and their coupled PCR assays. The software facilitates alignment of sequences surrounding SNVs and generates ligation probes while screening for dimerization potential. OLAgen successfully produced ligation probes that closely matched previously validated designs for HIV-1, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and KRAS, confirming its reliability and potential for clinical applications. The tool was used to generate new assays targeting Mycobacterium tuberculosis drug resistance and variants in the human JAK2, BRAF, and factor V genes, all of which demonstrated 100% sensitivity and specificity in controlled laboratory experiments. The OLAgen predicted assay designs detected mutant frequencies as low as 1% to 5% in wild-type backgrounds in proof-of-concept laboratory studies. OLAgen represents a significant advancement in accessible assay design, promoting the broader application of OLA technology in clinical and research settings.
To help guide treatment decisions and trial matching, tumor genomic profiling is an essential precision oncology tool. Liquid biopsy, a complementary approach to tissue testing, can assess tumor-specific DNA alterations...To help guide treatment decisions and trial matching, tumor genomic profiling is an essential precision oncology tool. Liquid biopsy, a complementary approach to tissue testing, can assess tumor-specific DNA alterations circulating in the blood. Labcorp Plasma Complete is a next-generation sequencing, cell-free DNA comprehensive genomic profiling test that identifies clinically relevant somatic variants across 521 genes in advanced and metastatic solid cancers. Over 800 unique sequencing libraries across 27 cancer types were evaluated to establish analytical sensitivity, specificity, accuracy, and precision, reproducibility, and repeatability (PRR). Sensitivity was verified for each variant type, with a median variant allele frequency (VAF) of 1.25% and 1.27% for panel-wide single nucleotide variants (SNVs) and insertions/deletions (indels) (sequence mutations), respectively, with <1% VAF sensitivity observed for clinically actionable variants, 1.72-fold for copy number amplifications (CNAs), 0.48% fusion read fraction for translocations, and 0.47% sequence mutation VAF for microsatellite instability-high (MSI-H). Specificity was 99.9999% for SNVs and 100% for other variant types. PRR resulted in 94.9% average positive agreement (APA) and 99.9% average negative agreement (ANA) for sequence mutations and 100% APA and ANA for CNAs, translocations, and MSI-H. Orthogonal assays were utilized to assess accuracy, demonstrating concordance of 97.4% positive percent agreement and >99.99997% negative percent agreement across all variants. Overall, the test demonstrates high sensitivity, specificity, accuracy, and robustness to enable informed clinical decision-making.
Paschal CR, Zalusky MPG, Beck AE
… +6 more, Gillentine MA, Narayanan J, Damaraju N, Goffena J, Storz SHR, Miller DE
J Mol Diagn
· 2025 Mar · PMID 39756651
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Current clinical testing approaches for individuals with suspected imprinting disorders are complex, often requiring multiple tests performed in a stepwise manner to make a precise molecular diagnosis. We investigated wh...Current clinical testing approaches for individuals with suspected imprinting disorders are complex, often requiring multiple tests performed in a stepwise manner to make a precise molecular diagnosis. We investigated whether whole-genome long-read sequencing could be used as a single data source to simultaneously evaluate copy number variants, single-nucleotide variants, structural variants, and differences in methylation in a cohort of individuals known to have either Prader-Willi or Angelman syndrome. Twenty-five individuals sequenced to an average depth of coverage of 36× on an Oxford Nanopore Technologies PromethION were evaluated. A custom one-page report was generated that could be used to assess copy number, single-nucleotide variants, and methylation patterns at select CpG sites within the 15q11.2-q13.1 region and prioritize candidate pathogenic variants in UBE3A. After training with three positive controls, three analysts blinded to the known clinical diagnosis arrived at the correct molecular diagnosis for 22 of 22 cases (20 true positive, 2 negative controls). Our findings demonstrate the utility of long-read sequencing as a single, comprehensive data source for complex clinical testing, offering potential benefits, such as reduced testing costs, increased diagnostic yield, and shorter turnaround times, in the clinical laboratory.
Roy S, Fussell AM, Jordan D
… +5 more, Kadri S, Leon A, Schmidt RJ, Temple-Smolkin RL, Merker JD
J Mol Diagn
· 2025 Jul · PMID 39734030
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The use of next-generation sequencing and other high-throughput technologies in the clinical molecular diagnostics laboratory requires the application of bioinformatics pipelines and other computational tools to analyze,...The use of next-generation sequencing and other high-throughput technologies in the clinical molecular diagnostics laboratory requires the application of bioinformatics pipelines and other computational tools to analyze, visualize, and store these clinical data. Clinical bioinformaticians, individuals with the skills to develop, validate, and deploy these tools in a clinical setting, are needed to ensure that these molecular diagnostic technologies can be appropriately used for clinical care. Building on existing expertise in informatics, next-generation sequencing, and clinical molecular diagnostics, the Association for Molecular Pathology has generated a series to establish an initial clinical bioinformatician body of knowledge. These articles cover the potential roles of the clinical bioinformatician, assist molecular laboratory and clinical bioinformatics directors in understanding the various roles of the clinical bioinformatics team members, and provide guidance regarding the competencies and skill sets required. The three articles within this Body of Knowledge cover the following knowledge cores: i) Molecular Diagnostics, ii) Clinical Bioinformatics, Software, and Database Knowledge, and iii) Clinical Laboratory Regulation and Data Security. Many of the topics covered in these articles are broad and rapidly evolving; therefore, this Association for Molecular Pathology Clinical Bioinformatician Body of Knowledge article series is designed to provide an initial framework for the core bioinformatics skills required to function successfully within a molecular diagnostic laboratory.
Consanguinity, prevalent in certain populations because of cultural and social factors, significantly increases the risk of genetic autosomal recessive disorders. In Lebanon, consanguineous marriages constitute 35.5% of...Consanguinity, prevalent in certain populations because of cultural and social factors, significantly increases the risk of genetic autosomal recessive disorders. In Lebanon, consanguineous marriages constitute 35.5% of unions, with first cousin marriages being the most common. This study aims to develop a model to predict consanguinity status using total runs of homozygosity (ROH) size derived from exome sequencing data. In this study, a cohort of 784 Lebanese individuals was analyzed, with consanguinity labels assigned based on pedigree information. ROHs were detected from exome sequencing data using AutoMap. The analysis focused on 521 subjects for whom the consanguinity or nonconsanguinity label was clearly determined, leading to the development of two logistic regression models: one including outliers (accuracy, 91%) and one excluding them (accuracy, 94%). The second model established specific ROH thresholds for categorizing consanguinity: nonconsanguineous [<40.28 megabases (Mb)], uncertain (40.28 to 79.17 Mb), probable consanguinity (79.18 to 118.06 Mb), and consanguineous (>118.06 Mb). This study provides a valuable tool for clinical genetics in populations with high consanguinity rates, offering insights into the genetic risks associated with consanguinity and aiding in the identification and counseling of affected individuals. Moreover, the current findings underline the importance of population-specific thresholds in accurately assessing consanguinity status.
J Mol Diagn
· 2025 Mar · PMID 39725012
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Pharmacogenetic-guided prescribing can lead to more accurate medicine selection and dosing, improving patient outcomes and leading to better use of health care budgets. Loss-of-function variants in CYP2C19 influence an i...Pharmacogenetic-guided prescribing can lead to more accurate medicine selection and dosing, improving patient outcomes and leading to better use of health care budgets. Loss-of-function variants in CYP2C19 influence an individual's ability to metabolize clopidogrel, increasing the risk of secondary vascular events following ischemic stroke and percutaneous coronary intervention. In acute clinical contexts, centralized laboratory-based testing is too slow to inform timely clinical decision-making. This work reports the development and analytical validation of the Genedrive CYP2C19 ID Kit, which provides rapid point-of-care genotyping from a buccal swab in approximately 1 hour. Buccal samples were collected from a total of 204 individuals between September 2023 and July 2024, alongside a blood or saliva sample for comparison with laboratory testing. In the final cohort of 202 patients, all point-of-care results were concordant with laboratory testing. In this assessment, the sensitivity and specificity of the CYP2C19 ID Kit was 100% (95% CI, 95.0%-100%) and 100% (95% CI, 97.2%-100%), respectively. The failure rate of the CYP2C19 ID Kit was 0.98%. This study confirms the analytical validity of the Genedrive CYP2C19 ID Kit. The Genedrive system is able to provide an accurate, rapid, noninvasive alternative to standard laboratory testing and can be used as a point-of-care test in the clinical environment.
Microsatellite instability (MSI) detection using tumor tissue is a well-established prognostic and predictive biomarker for certain types of cancers. However, tumor tissue samples are less convenient to obtain than blood...Microsatellite instability (MSI) detection using tumor tissue is a well-established prognostic and predictive biomarker for certain types of cancers. However, tumor tissue samples are less convenient to obtain than blood plasma samples. The main challenge facing next-generation sequencing-based MSI detection in blood plasma samples is the ultralow signal/noise ratio in plasma cell-free DNA (cfDNA). To address the challenge, plasmaMSI, a highly accurate cfDNA MSI detection method, is introduced with three novel performance-improving features: i) a set of stringent locus selection criteria to select loci with high robustness and compatibility across sequencing platforms; ii) a new deduplication strategy that greatly improves the signal/noise ratio for MSI detection; and iii) an MSI calling algorithm that customizes the baseline for each test sample based on its duplication rate. Through analytical validation in diluted cell line samples, the limit of detection of plasmaMSI was determined to be 0.15%. Furthermore, in analyzing 95 evaluable cfDNA samples from patients with gastrointestinal cancers, plasmaMSI exhibited a positive percentage agreement of 92.9% (39/42) and a negative percentage agreement of 100% (53/53) with tissue MSI-PCR. plasmaMSI provides novel solutions to key challenges in cfDNA MSI detection that have not been addressed by existing methods. It has also been systematically validated and is already used in clinical testing for patients with cancer.
Kirsten rat sarcoma viral oncogene homolog (KRAS) somatic mutations occur in 30% to 40% of patients with colorectal cancer (CRC). These were thought to equally affect prognosis and resistance to anti-epidermal growth fac...Kirsten rat sarcoma viral oncogene homolog (KRAS) somatic mutations occur in 30% to 40% of patients with colorectal cancer (CRC). These were thought to equally affect prognosis and resistance to anti-epidermal growth factor receptor agents; however, recent data show the activity of KRAS-G12C and pan-RAS inhibitors. The effects of uncommon KRAS (uKRAS) variants are largely unexplored. The distribution and pathogenicity of uKRAS mutations and their relationship with patients' clinicopathologic features were assessed. A total of 2427 CRCs were profiled for KRAS using next-generation sequencing (NGS). The study and control groups included patients with uKRAS (<1% frequency in CRC data sets on cBioPortal) and canonical KRAS mutations, respectively. In silico protein structure modifications and prediction analyses were performed by using PyMOL, trRosetta, and PolyPhen-2. uKRAS mutations affected 35 cases (1.5%), with G13C (28.6%), G12R (20%), and V14I (8.6%) being most common. Missense mutations (D33E, G12W, G12F, Q22H, Q61L, and L19F) occurred in nine cases (25.7%). Duplications (G10dup and L52_G60dup) affected two cases. Pathogenicity analyses showed that G12W, Q22R, L56V, and A130I mutations are probably damaging, with scores between 0.928 and 1.000. No differences were seen in clinicopathologic features. uKRAS mutants had lower event-free survival but no difference in overall survival compared with controls. Although these data are hypothesis generating and need further confirmation, they highlight the importance of NGS-based profiling to identify CRC patients with uKRAS mutations as candidates for personalized therapy.
Poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors represent a significant advancement in the treatment of epithelial ovarian cancer, triple-negative breast cancer, pancreatic cancer, and castrate-resistant...Poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors represent a significant advancement in the treatment of epithelial ovarian cancer, triple-negative breast cancer, pancreatic cancer, and castrate-resistant prostate cancer, and they are poised to improve treatment in an increasing number of other cancer types. PARP inhibitor efficacy as monotherapy has been primarily observed in tumors with deleterious genetic variants in genes involved in the homologous recombination repair pathway. Tumors without these variants have also been shown to respond; notably, those with hypermethylation at all alleles of the BRCA1 or RAD51C promoter can respond to PARP inhibitors. These epigenetic biomarkers therefore represent a patient population that may also benefit from this targeted therapy. However, no robust test has been conducted to identify these biomarkers in routine clinical specimens that is amenable to implementation for decentralized testing. This study describes the analytical and clinical validation of a BRCA1 and RAD51C promoter methylation test that can be run with a single-day library preparation workflow for sequencing on any next-generation sequencing platform. The results show that this test can accurately quantitate the level of promoter methylation at the BRCA1 and RAD51C genes using formalin-fixed, paraffin-embedded samples, even when the extracted DNA is extremely degraded or the input amount is limited. This test increases the precision of diagnostic tests aimed at identifying patients who are likely and unlikely to respond to PARP inhibitor therapy.
Ball M, Romanovsky E, Schnecko F
… +16 more, Kirchner M, Neumann O, Brandt R, Beck S, Seker-Cin H, Kluck K, Ourailidis I, Goldschmid H, Fink A, Volckmar AL, Menzel M, Allgäuer M, Schirmacher P, Budczies J, Stenzinger A, Kazdal D
J Mol Diagn
· 2025 Feb · PMID 39674366
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The adoption of comprehensive genomic profiling in oncology has rapidly increased the demand for standardized tumor sample processing in diagnostic laboratories. Automation of DNA and RNA library preparation workflows of...The adoption of comprehensive genomic profiling in oncology has rapidly increased the demand for standardized tumor sample processing in diagnostic laboratories. Automation of DNA and RNA library preparation workflows offers the possibility to scale-up and standardize sample processing. We report on the clinical implementation of the automated TruSight Oncology 500 High-Throughput library preparation workflow from formalin-fixed, paraffin-embedded tumor samples using the Biomek i7 hybrid Workstation. Using the same input amount, the automated workflow was validated against manual library preparation. Quality control metrics (total and mapped reads, median insert size, and median exon coverage) and the detection of tumor mutational burden, a complex biomarker, were concordant between the manual and automated workflows. The automated workflow was implemented on a total of 2997 pan-cancer clinical samples to detect genomic variants and complex biomarkers. Workflow automation resulted in a 4-fold reduction in hands-on time and a 1.7-fold reduction in total runtime compared with manual library preparation (6 hours vs. 23 hours; 24 hours vs. 42.5 hours, respectively) for a 48 DNA + 48 RNA sample batch. The automated workflow required one technician versus three technicians to manually prepare the same number of libraries. This study shows that implementation of the automated TruSight Oncology 500 High-Throughput workflow significantly reduced hands-on time and processing time per sample compared with manual library preparation.
Di J, Sheng T, Arora R
… +5 more, Stocks-Candelaria J, Wei S, Lutz C, Yalniz FF, Zhang S
J Mol Diagn
· 2025 Feb · PMID 39615653
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Accurate monitoring of minimal residual disease (MRD) is crucial for effective management of patients with acute myeloid leukemia (AML). This study aims to validate MRD detection of the seven most common IDH1 and IDH2 mu...Accurate monitoring of minimal residual disease (MRD) is crucial for effective management of patients with acute myeloid leukemia (AML). This study aims to validate MRD detection of the seven most common IDH1 and IDH2 mutations in patients with AML using a QuantStudio 3D digital PCR platform. This assay demonstrated a high concordance for the variant allele frequencies between digital PCR and next-generation sequencing assays. Precision analysis revealed only small variation (<0.5 log10) for all mutations near or at the limit of detection level. This validation also showed a great reproducibility for interrun and intrarun comparisons (28 runs, variation ranges from 0 to 0.48 log10), ensuring comparable results for patient follow-ups. The limit of detection was determined to be 0.1% for all mutations, except the IDH2 R140Q mutation, which was 0.5%. Controls and acceptable ranges were also established for each mutation during validation. This study suggests that the QuantStudio 3D digital PCR assay is a quantitative, sensitive, and reproducible platform for monitoring MRD in patients with AML.
BCR::ABL1 digital PCR is a promising technique for the quantification of deep molecular responses (DMRs) in chronic myeloid leukemia. It provides an improved precision and sensitivity compared with conventional real-time...BCR::ABL1 digital PCR is a promising technique for the quantification of deep molecular responses (DMRs) in chronic myeloid leukemia. It provides an improved precision and sensitivity compared with conventional real-time quantitative PCR (qPCR), which is particularly relevant in the context of prediction of successful treatment-free remission. This study assessed the feasibility of BCR::ABL1 digital PCR in clinical practice. A total of 168 DMR samples of patients with chronic myeloid leukemia aiming for a treatment-free remission attempt were assessed by both digital PCR and qPCR. Digital PCR was performed with the droplet-based Bio-Rad QXDx BCR-ABL %IS assay, using eight replicates per sample. qPCR was performed with the fully automized Cepheid Xpert BCR-ABL Ultra assay. Various technical and practical aspects of BCR::ABL1 quantification using digital PCR were assessed. The reported limit of detection of the qPCR is molecular response 4.5, requiring an equivalent of 32,000 ABL1 transcripts. Using digital PCR, a median number of ABL1 of approximately 300,000 were obtained. BCR::ABL1 was quantifiable by digital PCR in 68% of the samples below qPCR's limit of detection. In addition, e13a2 and e14a2 BCR::ABL1 transcript types could be discriminated based on the mean fluorescence intensity of BCR::ABL1-positive droplets. BCR::ABL1 digital PCR is feasible for DMR quantification in clinical practice and offers an increased sensitivity over qPCR.
Chimerism test was evaluated to predict leukemia relapse. Increasing mixed chimerism (IMC), defined as recipient increase ≥0.1% in peripheral blood total cell chimerism, was used as a surrogate of disease activity. Combi...Chimerism test was evaluated to predict leukemia relapse. Increasing mixed chimerism (IMC), defined as recipient increase ≥0.1% in peripheral blood total cell chimerism, was used as a surrogate of disease activity. Combination of quantitative PCR and short-tandem repeat method was applied to achieve high assay sensitivity. Total of 184 patients received stem cell transplant for acute myeloid leukemia (N = 110), acute lymphocytic leukemia (N = 41), myelodysplastic syndrome (N = 30), and 2389 chimerism tests (median follow-up, 1054 days). Sixty-six patients relapsed, and 118 patients did not. Cumulative incidence of relapse increased after 1 IMC or ≥2 consecutive IMCs (hazard ratios, 9.9 and 44.4, respectively). Predicted percentage relapse by day 30 after IMC was 0% (0 IMC), 10% (1 IMC), and 40% (≥2 IMCs). The last chimerism results before relapse detected IMC in 57 of 66 relapsed patients (sensitivity, 86.4%). Nine patients had no IMC before relapse (false negative) because of rapidly evolving relapse (N = 4) or extramural relapse (N = 5). In 118 patients without relapse, 158 of 1873 tests detected IMC (false positive, 8.4%; specificity, 91.6%). The false-positive rates increased with higher percentage recipient T-cell chimerism levels, indicating T-cell contamination as a cause. Chimerism monitoring predicts relapse. However, caution must be taken for false-positive or false-negative IMCs. T-cell removal can improve chimerism test specificity in patients with mixed T-cell chimerism.
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) application for targeted sequencing has made a breakthrough in the genomic research era. High diversity in the capsula...Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) application for targeted sequencing has made a breakthrough in the genomic research era. High diversity in the capsular polysaccharide (cps) locus of Streptococcus pneumoniae has hampered identification of the serotype. This study developed a new serotyping method for S. pneumoniae using CRISPR/Cas9-targeted sequencing with the Oxford Nanopore Technologies platform. A probe was designed at the position of the cps locus using an excision approach on two sides flanking genes between the dexB and aliA genes with approximately 20 kb. A native barcoding method was used for multiplexing. The probe will attach to a specific side followed by attachment of CRISPR/Cas9 to cut the recognition area. The study used de novo assembly to reconstruct sequence reads, which were analyzed using PneumoCRISPR, a new serotyping pipeline for Oxford Nanopore Technologies sequencing data output. Four CRISPR/Cas9 probes have been designed and recognize the cps locus of S. pneumoniae. Serotyping results align precisely with serotyping data from whole-genome sequencing. This serotyping method also allows researchers to use multiple samples in a single run. The new serotyping method based on CRISPR/Cas9-targeted sequencing holds immense promise for serotype identification of S. pneumoniae.