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PubMed2026

Applications of artificial intelligence in robot-assisted surgery: a systematic review.

To characterize applications of artificial intelligence (AI) in robot-assisted surgery, summarize technical and clinical performance, and assess the quality of the available evidence. PubMed, Web of Science Core Collection, and Scopus were searched for English-language journal articles published from 1 January 2020 through 31 October 2025. Randomized, observational, model-development, validation, and feasibility studies evaluating AI in robot-assisted surgery or closely related image-guided minimally invasive workflows were eligible. Two reviewers independently performed study selection, data extraction, and risk-of-bias assessment. Owing to heterogeneity in surgical procedures, AI tasks, analytical units, validation strategies, and outcomes, findings were synthesized descriptively without statistical pooling. The review was registered in the International Prospective Register of Systematic Reviews (CRD420251175699). Seventeen studies were included: seven clinical prediction or decision-support studies, eight intraoperative recognition, segmentation, or image-guided studies, and two training or workflow studies. Five prediction studies reported area-under-the-curve values of 0.74-0.95. Technical studies reported F1 or Dice scores of 0.525-0.995 and task-specific accuracies of 0.840-0.998. Two randomized studies suggested benefits for personalized suturing feedback and automated camera control, but neither established improved patient outcomes. Only one study had low overall risk of bias; the remaining studies were at high or unclear risk or raised some concerns. AI applications in robot-assisted surgery show promise for prediction, intraoperative perception, training, and workflow support. Evidence primarily demonstrates technical feasibility rather than established clinical effectiveness. Independent multicenter validation and prospective evaluation of patient, educational, and workflow outcomes are required before widespread implementation.

Robotic Surgical Procedures↗

PubMed2026

AI echo INSIGHT study: A prospective blinded randomized trial of artificial intelligence echocardiogram interpretation.

BACKGROUND: Transthoracic echocardiography (TTE) is the most commonly performed cardiac imaging modality with over 30 million studies annually. Demand for timely expert interpretation continues to outpace capacity, creating diagnostic delays and inter-observer variability that impact patient care. Recent research has suggested computer vision artificial intelligence (AI) models can generate accurate preliminary comprehensive TTE reports, however, prospective evaluation is needed to determine whether AI-assisted TTE interpretation can improve clinician efficiency while preserving diagnostic accuracy. METHODS: AI ECHO INSIGHT is a prospective randomized blinded clinical trial conducted at Kaiser Permanente Northern California that will evaluate 1200 historical TTE studies (1000 consecutive unselected studies plus 200 with moderate or greater valvular disease) interpreted using three workflows: (1) AI-generated preliminary report finalized by a blinded cardiologist (AI-assisted); (2) cardiologist-generated preliminary report finalized by a blinded cardiologist (cardiologist-assisted); and (3) sonographer-generated preliminary report finalized by a blinded cardiologist (sonographer-assisted). The primary outcome is the rate of substantial change between preliminary and final reports, comparing the AI-assisted workflow to the pooled cardiologist-assisted and sonographer-assisted workflows. Secondary outcomes include cardiologist interpretation time for report finalization, superiority testing for diagnostic accuracy, and reporting consistency. CONCLUSION: AI ECHO INSIGHT is a prospective randomized blinded clinical trial evaluating the clinical impact of AI-assisted TTE interpretation on diagnostic accuracy, cardiologist efficiency, and reporting consistency in real-world echocardiography workflows. TRIAL REGISTRATION: ClinicalTrials.gov registration number NCT07229300.

Humans↗

PubMed2026

A hybrid and cost-efficient barcoding strategy for full-length 16S rRNA gene nanopore sequencing of environmental samples.

BACKGROUND: Accurate species-level identification of bacteria in complex environmental samples is essential for applications in biotechnology, ecological monitoring, and clinical diagnostics. Short-read platforms such as Illumina frequently truncate the 16S rRNA gene, limiting taxonomic resolution. In this work, we applied Oxford Nanopore Technology (ONT) long-read sequencing to full-length 16S rRNA amplicon in samples from natural soil amended with lignocellulosic biomass and a simplified microbial community derived from cultures grown on selective and differential carboxymethyl cellulose (CMC)-based substrates, with the aim to evaluate the difference in performance between a real, complex community and a less complex system. To reduce consumable costs, we substituted the standard ONT Barcoding kits with an in-house hybrid barcoding workflow. Specifically, PacBio PCR-based barcoding protocol was used for sample indexing, followed by library preparation using the ONT Ligation Sequencing Kit. This simplified approach retained compatibility with MinION and Flongle flow cells and supported accurate downstream demultiplexing while lowering barcode costs substantially. Additionally, a new bioinformatic workflow tailored to ONT data was implemented. RESULTS: Overall, the hybrid protocol significantly reduced per-sample barcoding costs while preserving high sequencing quality and throughput. The sequencing run yielded over 5 Gb of quality-filtered data (Q-score ≥ 10). Furthermore, the new bioinformatic workflow allowed taxonomic assignment at the species level for 49.38% of annotated taxa, compared to just 4.59% using Illumina NovaSeq sequencing of the V3-V4 region. ONT also recovered 2.3 times more genera and 1.3 times more families. Although 16S rRNA gene sequencing often cannot distinguish between closely related species, particularly within taxonomically complex groups, in this work, full-length reads substantially improved both taxonomic resolution and database matching. CONCLUSIONS: These results show that full-length 16S rRNA sequencing with ONT, paired with a low-cost barcoding strategy, enhanced taxonomic resolution compared to short-read workflows. This approach also offers a scalable and cost-effective option for high-resolution microbiome profiling in research and applied settings.

RNA, Ribosomal, 16S↗

PubMed2026

Clinical accuracy and short-term outcomes of intraoral photogrammetry for complete-arch implant rehabilitation: A retrospective multicentre study on 35 patients.

OBJECTIVES: To evaluate the clinical accuracy and short-term outcomes of complete-arch implant-supported fixed dental prostheses (ISFDPs) fabricated using an intraoral photogrammetry (IPG) based digital workflow in completely edentulous patients. METHODS: This multicenter retrospective clinical study included 35 patients rehabilitated with 52 complete-arch ISFDPs (10 FP1, 18 FP2 and 24 FP3 restorations) supported by 221 implants. All definitive prostheses were designed and fabricated using a fully digital workflow initiated by IPG acquisition with the Aoralscan Elite IPG® (SHINING 3D). The primary outcome was clinical accuracy, assessed at definitive prosthesis delivery through evaluation of passive fit using the Sheffield test and radiographic verification. Secondary outcomes included biologic and prosthetic complications, as well as implant and prosthesis survival rates during the follow-up. RESULTS: Passive fit was achieved in all definitive restorations (100%). Radiographic evaluation confirmed accurate marginal adaptation at the implant-prosthesis interface in all cases. No statistically significant differences in clinical accuracy were observed according to treated arch, number of supporting implants, or prosthetic design (P > .05). During a mean follow-up period of 12.1 ± 3.5 months, biologic and prosthetic complications were limited and generally minor. Implant survival was 99.5%, and prosthesis survival was 100%. CONCLUSIONS: Within the limitations of this retrospective clinical study, the IPG based workflow demonstrated high clinical accuracy and predictable short-term outcomes for complete-arch implant rehabilitation, consistently enabling passive fit and favorable prosthetic performance. CLINICAL RELEVANCE: IPG may represent a clinically reliable and predictable approach for complete-arch digital implant impression acquisition. The high rates of passive fit, together with the low incidence of biologic and prosthetic complications observed in this multicenter clinical study, support the use of IPG based workflows for the fabrication of complete-arch ISFDPs.

Humans↗

PubMed2026

Rapid CRISPR-based bovine embryo sexing to streamline genotype-informed cattle breeding.

Cattle in vitro fertilisation and embryo transfer programmes increasingly rely on embryo-level selection to accelerate genetic gain, but current sexing and genotyping workflows can be costly, slow and logistically demanding. This study developed an efficient, low-resource workflow for bovine embryo sexing that combines whole genome amplification (WGA) with recombinase polymerase amplification-CRISPR-Cas12a (RPA-Cas12a). It also assessed whether the same WGA biopsy products could be used for downstream single nucleotide polymorphism (SNP) microarray genotyping. A one-tube RPA-Cas12a assay targeting the bovine Y-chromosome S4 repeat was developed for fluorescence and lateral flow assay (LFA) readouts. Analytical sensitivity was assessed using serially diluted bovine genomic DNA (gDNA), and breed robustness was tested using male and female gDNA from five major beef breeds and Holstein cattle. The workflow was then applied to WGA products from 22 bovine blastocyst biopsies, with sex calls validated against an established real-time PCR melt curve assay and 100K SNP microarray genotyping. The assay detected male bovine gDNA down to 100 pg using both fluorescence and LFA readouts, with no signal from female gDNA. Male-specific detection was consistent across all breeds tested. All WGA-RPA-Cas12a sex calls from blastocyst biopsies were concordant with real-time PCR and SNP microarray sex calls, and WGA biopsy products produced genome-wide SNP call rates above 85%. This workflow provides a practical approach for rapid bovine embryo sex triage and could reduce unnecessary cryopreservation and genotyping while improving the efficiency of genotype-informed cattle breeding programmes.

Bovine embryo↗

PubMed2026

Liquid biopsy-based detection of circulating and exfoliated cholangiocarcinoma tumor cells from blood and bile using heparan sulfate octasaccharides on integrated microfluidic systems.

Early diagnosis of cholangiocarcinoma (CCA) remains challenging because existing diagnostic approaches often lack sufficient sensitivity for reliable detection of early-stage disease. Circulating tumor cells (CTCs) in blood and exfoliated tumor cells (ETCs) in bile represent valuable targets for liquid biopsy-based detection; however, their low abundance and the complexity of clinical sample analysis pose substantial technical challenges for reliable enrichment and identification. Herein, we present a reproducible workflow for isolating and identifying CCA tumor cells from blood for CTCs and bile for ETCs using synthetic cell-surface heparan sulfate (HS) octasaccharide-functionalized magnetic beads (MBs) on integrated microfluidic systems. The method combined sample pre-processing, magnetic bead-based enrichment, controlled low-shear mixing and immunofluorescence-based identification into a unified workflow compatible with distinct clinical sample types. Key operational parameters, including MB concentration, mixing frequency, and pressure settings, were detailed to facilitate consistent performance. Using this workflow, tumor cell capture rates of approximately 70% in bile (for ETCs) and blood (for CTCs) were achieved, with a total processing time of 60-90 min per sample under clinically relevant low-abundance conditions. The platform enables reliable detection of as few as 1 tumor cell per mL of blood or bile. This method provides a practical and adaptable strategy for glycosaminoglycan-mediated liquid biopsy applications and may be extended to other tumor-cell enrichment workflows involving heterogeneous cell-surface interactions.

Humans↗

PubMed2026

Evaluation and Optimization of Different Digestion Strategies for In-Depth Proteomic Characterization of Residual Host Cell Proteins in rAAV-Based Gene Therapy Products.

Recombinant adeno-associated virus vectors (rAAVs) are the most important vectors for in vivo gene therapies, yet their safety relies on low levels of residual host cell proteins (HCPs). While mass spectrometry-based proteomics enables sensitive and untargeted HCP profiling, it faces challenges with matrix interferences from purification buffers and the high dynamic range between abundant viral capsids and trace HCPs. Although sample preparation methods that address these challenges are well-established for antibody products, their adaptation to rAAV purification stages and products remains largely unexplored. In this study, we systematically evaluated three widely used proteomic sample preparation workflows─In-Solution, FASP, and SP3─across different stages of rAAV purification. In addition, each workflow was tested under both standard denatured digestion conditions and a "native" digestion strategy designed to reduce dynamic range by preserving capsid integrity while selectively digesting HCPs. This comparison identified the native FASP protocol as the most effective sample preparation method for overcoming matrix interference and dynamic range challenges, consistently outperforming other workflows in HCP identification across purification stages. Further optimization of the native FASP workflow enhanced its performance, achieving the highest host cell (HC) proteome depth, particularly in highly purified drug substance samples. This optimized sample preparation strategy provides a robust and easy-to-use framework for deep characterization of the host cell proteome in rAAV samples. By enabling deeper insights into the HCP profile, this approach supports improved understanding of the rAAV purification process and facilitates the development of targeted strategies to enhance product quality and safety.

Dependovirus↗

PubMed2026

Selective Enrichment of Newly Synthesized Proteins Using Phos-Tag Click Tip Enables Nascent Proteome Analysis in Influenza A Virus Infection.

Profiling of newly synthesized proteins (NSPs) provides access to dynamic changes in protein production that accompany acute cellular responses. Bioorthogonal noncanonical amino acid tagging (BONCAT)-based approaches enable selective labeling of NSPs; however, their broader application remains constrained by labor-intensive enrichment workflows and limited sensitivity for direct peptide-level analysis. Here, we developed a workflow termed "Phos-tag Click Tip" by integrating a phosphorylated variant of bicyclononyne (pBCN) with Phos-tag affinity purification to selectively capture azidohomoalanine (AHA)-labeled peptides for newly synthesized proteome analysis (NSProteomics). This approach overcomes key limitations of conventional proteomics and BONCAT-based strategies by enabling efficient enrichment and sensitive detection of NSP-derived peptides. Using this workflow, we performed comprehensive NSP profiling of host cells during influenza A virus infection. We identified dynamic changes in distinct NSP profiles associated with viral replication, host restriction, and immune responses, many of which were not readily detected with conventional whole-cell- or phospho-proteomic analyses. Overall, the Phos-tag Click Tip workflow provides a complementary approach for stimulus-responsive NSP profiling, offering functionally relevant insights into host-virus interactions and cellular response mechanisms.

Proteome↗

PubMed2026

StrainMake: reproducible hybrid metagenomics with MAG recovery and strain-level resolution.

SUMMARY: Metagenomic workflows involve complex multi-step analyses, from quality control and assembly to binning, annotation, and strain-level profiling. Few existing metagenomic pipelines achieve the combination of flexibility, reproducibility, and hybrid assembly support within a unified workflow. We present StrainMake, a Snakemake-based workflow for de novo metagenomic analysis from short, long, or hybrid sequencing data. StrainMake integrates widely used tools across all major steps-quality control, assembly, binning, dereplication, taxonomic and functional annotation-while also providing non-redundant gene catalogues, community-scale metabolic models, and strain-level microdiversity metrics. The modular design enables the use of alternative tools, scalable execution on HPC systems, and full reproducibility through Snakemake and Conda. RESULTS: Applied to the CAMI II strain-madness dataset, StrainMake produced high-quality assemblies and metagenome-assembled genomes (MAGs), while enabling strain-resolved comparisons across samples. Hybrid assemblies improved contiguity, whereas short-read assemblies offered faster runtimes, illustrating the workflow's benchmarking capacity. AVAILABILITY AND IMPLEMENTATION: StrainMake is open source and available at https://github.com/UMMISCO/strainmake, together with comprehensive documentation. Generated data are deposited in Zenodo (doi: 10.5281/zenodo.16950162).

Metagenomics↗

PubMed2026

Genetic testing and reporting: What the endocrinologists should know and can expect (Joint position paper of the ENDO-ERN).

Over the last decade, next generation sequencing (NGS) has become an essential tool for diagnostic DNA testing in human genetics. To improve the understanding of available genetic testing strategies and to facilitate the request of genetic testing in daily endocrine practice, clinical and laboratory experts in the field have summarized the major issues which should be known and considered. In this joint position paper of the ENDO-ERN, the roles and responsibilities of the health care professionals involved in the diagnostic workflow are described, and the major issues concerning genetic testing workflows are overviewed. These issues encompass all relevant steps, including test request and pre-analytical procedures, laboratory and data processing workflows, quality assurance, and reporting. As NGS procedures result in an increasing number of variants of unknown significance and incidental findings, these aspects are addressed as well. Accompanied by illustrations of the genetic diagnostic workflow and of concise reports for a fast orientation about the major aspects of genetic testing, this joint paper should support the health care professionals during a request for genetic testing.

VUS↗

PubMed2026

Post-Hoc Long-Read Sequencing Links Leukemic Mutation Status to Single-Cell Transcriptomes.

Single-cell RNA-sequencing-based characterization of cells that belong to the neoplastic clone is a major challenge in hematologic neoplasms, where malignant and normal cells coexist. Confident molecular profiling requires simultaneous analysis of gene expression and genetic mutations in individual cells, an ability that is not supported by the standard 10X Genomics workflow. Here, we systematically evaluated the potential and limitations of repurposing amplified cDNA generated during the 10X Genomics 3' workflow for post hoc genotyping of individual cells. We first established a mixed leukemic cell line system comprising one cell line with KIT point mutations and another with the BCR::ABL1 fusion gene. Targeted long-read PacBio sequencing enabled post hoc assignment of mutation data to transcriptionally profiled cells, but recovery differed between targets. Consistent with ambient RNA in microfluidics-based single-cell workflows, mutation-associated transcripts were detected in cells not expected to carry the corresponding mutations, illustrating how transcript recovery complicates cell-level genotype assignment. Target-specific thresholds mitigated this source of misclassification. In primary chronic myeloid leukemia samples, the post hoc approach detected BCR::ABL1-positive cells at diagnosis, but not during imatinib treatment. Together, we present a framework for adding mutation status to cells already profiled using the 10X Genomics workflow and highlight broader considerations for transcript-based single-cell genotyping.

BCR::ABL1↗

PubMed2026

Applicability of Nanopore-only whole-genome sequencing for Pseudomonas aeruginosa outbreak investigation in the ICU setting: a multicentric study.

UNLABELLED: Pseudomonas aeruginosa outbreaks frequently occur in intensive care units (ICUs). In particular, ICU patients requiring mechanical ventilation are vulnerable to P. aeruginosa ventilator-associated pneumonia, which is associated with high morbidity and mortality. Fast and accurate genotyping during the early stage is crucial to document and manage P. aeruginosa outbreaks at the ICU. In this study, we have evaluated the applicability of Oxford Nanopore whole-genome sequencing (WGS) for outbreak investigation and antimicrobial resistance (AMR) prediction. To evaluate whether a Nanopore-only WGS workflow was able to reproduce Illumina-confirmed transmission clusters, 19 P. aeruginosa isolates from ICUs at UZ Brussels (Belgium) that were previously sequenced with Illumina were sequenced using a Nanopore-only workflow based on the latest V14 chemistry, followed by bioinformatic analysis via BugSeq and MBioSEQ Ridom Typer. Although both bioinformatic platforms showed high concordance between Illumina and Nanopore data, MBioSEQ Ridom Typer yielded the lowest allelic distance (maximum one cgMLST allele), confirming all outbreak clusters. When applying the Nanopore-only workflow to longitudinally collected isolates, low genetic heterogeneity (maximum three cgMLST alleles) was observed between isolates from the same patient. WGS and subsequent outbreak analysis of 65 respiratory P. aeruginosa isolates collected from 38 different ICU patients across six Belgian hospitals during a 9-month period showed no intra- or inter-hospital transmission. When the Nanopore-only WGS data were used to predict AMR, there was high categorical agreement (95%) between AMR genotype and phenotype. These findings highlight the potential of Nanopore WGS as a rapid and accurate tool for outbreak investigation of P. aeruginosa. IMPORTANCE: In recent years, Nanopore sequencing has found its way to clinical laboratories because of its affordability, scalability, and, most importantly, its ability to obtain sequencing results in near-real time. However, despite improved raw read accuracies with the latest generation R10.4.1 flow cells, the question remains whether the achieved accuracy is sufficient for accurate bacterial outbreak investigation, particularly in high-risk settings such as intensive care units (ICUs). In this study, we show that Nanopore-only whole-genome sequencing (WGS) is able to match Illumina-only WGS in terms of accuracy for Pseudomonas aeruginosa outbreak investigation in the ICU setting, although important sequence type-dependent and even strain-specific methylation issues need to be resolved in order to guarantee this accuracy. By providing a fast and accurate workflow for reliable P. aeruginosa outbreak investigation, this study could pave the way for large-scale implementation of Nanopore-only WGS, leading to faster outbreak response times.

Humans↗

PubMed2026

pmultiqc: An Open-Source, Lightweight, and Metadata-Oriented QC Reporting Library for MS Proteomics.

The increasing scale and complexity of proteomics data demand robust, scalable, and interpretable quality control (QC) frameworks to ensure data reliability and reproducibility. Here, we present pmultiqc, an open-source Python package that standardizes and generates web-based QC reports across multiple proteomics data analysis platforms. Built on top of the widely adopted MultiQC framework, pmultiqc offers specialized modules tailored to mass spectrometry workflows, with full initial support for quantms, DIA-NN, MaxQuant/MaxDIA, FragPipe, and mzIdentML/mzML-based pipelines. The package computes a wide range of QC metrics, including raw intensity distributions, identification rates, retention time consistency, and missing value patterns, and presents them in interactive, publication-ready reports. By leveraging sample metadata in the Sample and Data Relationship Format format, pmultiqc enables metadata-aware QC and introduces, for the first time in proteomics, QC reports and metrics guided by standardized sample metadata. Its modular architecture allows easy extension to new workflows and formats. Alongside comprehensive documentation and examples for running pmultiqc locally or integrated into existing workflows, we offer a cloud-based service that enables users to generate QC reports from their own data or public PRIDE datasets.

Proteomics↗

PubMed2026

Systematic performance evaluation and application validation of an end-to-end NGS workstation.

Next-generation sequencing (NGS) library preparation is a core component of precision genomics, but it is commonly constrained by inefficiency, variability, and low throughput of manual protocols. To address these limitations, we developed and systematically evaluated a fully automated NGS workstations and further validated its performance across representative application scenarios. The automated system reduced total processing time from 8 to 10 to 4–6 h. At the same time, it maintained similar performance in pre-library metric, including DNA yield and fragment size, as well as post-capture sequencing metrics (Q30 > 90%, mapping rates > 95%, on-target rates 85–90%). The duplication rate was reduced to 5–8%, compared with 10–15% for manual methods, indicating increased library complexity. Bioinformatic evaluation of inter-species read mapping showed minimal cross-contamination, with a maximum contamination ratio of 0.0003%, indicating effective sample isolation in the automated workflow. High concordance in variant detection was observed between automated and manual workflows. Overall, this automated workstation provides a standardized and reproducible workflow that supports scalable precision genomics applications.

High-Throughput Nucleotide Sequencing↗

PubMed2026

Determinants of nucleic acid quality in formalin-fixed paraffin-embedded bladder urothelial carcinoma specimens: effects of specimen type, fixation conditions, and storage time.

Formalin-fixed paraffin-embedded (FFPE) tissue is the principal substrate for cancer genomic profiling, yet pre-analytical factors affect nucleic acid quality. In our workflow, transurethral resection (TUR) specimens of bladder urothelial carcinoma are fixed promptly but exposed to electrocautery, whereas robot-assisted radical cystectomy specimens are refrigerated overnight before fixation; we compared nucleic acid quality between these specimen types. We analyzed 56 FFPE primary bladder urothelial carcinoma specimens (33 TUR, 23 cystectomy). DNA quality was assessed by the DNA Integrity Number (DIN) and short-to-long cycle threshold ratio (S/L Ct ratio), and RNA quality by the RNA Integrity Number (RIN) and percentage of RNA fragments ≥200 nucleotides (DV200), with group comparisons, Spearman correlations, and multivariable regression (specimen type, fixation duration, storage time). The median DIN (4.4 vs. 3.8, p = 0.004) and S/L Ct ratio (0.943 vs. 0.894, p < 0.001) were higher in TUR than cystectomy specimens; the RIN was also higher (1.9 vs. 1.7, p = 0.040), though low in both groups, and DV200 did not differ (50.3% vs. 47.7%, p = 0.934). In multivariable analysis, TUR specimen type independently predicted higher DIN (β = +0.460, p = 0.040) and S/L Ct ratio (β = +0.442, p = 0.009), whereas longer storage time independently decreased the S/L Ct ratio (β = -0.513, p < 0.001) and RIN (β = -0.352, p = 0.010). In our institutional workflow, TUR specimens showed higher DNA quality metrics than robot-assisted radical cystectomy specimens subjected to overnight refrigerated pre-fixation delay, whereas this workflow-associated advantage did not clearly extend to DV200-defined RNA fragment-length quality. Storage time was associated with selected aspects of nucleic acid deterioration.

Humans↗

PubMed2026

Accelerating Lung Cancer Management Through Previsit Liquid Biopsy: Results From the LUNG-FAST Pilot Study.

BACKGROUND: Timely molecular profiling is essential for treatment selection in non-small cell lung cancer (NSCLC), yet delays in biomarker testing remain common. We evaluated the feasibility and early clinical impact of a nurse navigator-driven workflow to initiate liquid biopsy before the initial oncology visit. METHODS: LUNG-FAST (Liquid Biopsy for Urgent Neoplastic Genomic Profiling Focused Accelerated Stratification and Testing) was a 4-month prospective pilot at a tertiary cancer center. Intake nurse navigators identified eligible patients with suspected or newly diagnosed lung cancer and facilitated previsit liquid biopsy ordering. Feasibility, turnaround times, genomic findings, and early clinical outcomes were assessed. RESULTS: Among 64 patients, intake nurse navigators identified 94% (60/64) of eligible cases. Liquid biopsy was ordered in 58 patients, with 62% (36/58) placed before the initial oncology visit. Median turnaround time from blood draw to results was 8.5 days for commercial testing and 12.5 days for institutional testing. FDA-actionable genomic alterations were identified in 34% (22/64) of patients, while an additional 11% (7/64) harbored clinically relevant, non-FDA-actionable alterations. Overall, FDA-actionable or clinically relevant alterations were identified in 45% (29/64), with 22% detected by liquid biopsy and an additional 23% by tissue-only profiling. Median time from new patient visit to systemic therapy was 26 days. CONCLUSIONS: A nurse navigator-driven workflow enabling previsit liquid biopsy is feasible and identifies actionable genomic alterations in a substantial proportion of patients with lung cancer. Plasma and tissue profiling are complementary, and earlier plasma-based testing may expedite treatment decision-making while highlighting opportunities to optimize biomarker testing workflows.

Humans↗

PubMed2020

Genomic Analysis of Circulating Tumor Cells at the Single-Cell Level.

Circulating tumor cells (CTCs) have a great potential for noninvasive diagnosis and real-time monitoring of cancer. A comprehensive evaluation of four whole genome amplification (WGA)/next-generation sequencing workflows for genomic analysis of single CTCs, including PCR-based (GenomePlex and Ampli1), multiple displacement amplification (Repli-g), and hybrid PCR- and multiple displacement amplification-based [multiple annealing and loop-based amplification cycling (MALBAC)] is reported herein. To demonstrate clinical utilities, copy number variations (CNVs) in single CTCs isolated from four patients with squamous non-small-cell lung cancer were profiled. Results indicate that MALBAC and Repli-g WGA have significantly broader genomic coverage compared with GenomePlex and Ampli1. Furthermore, MALBAC coupled with low-pass whole genome sequencing has better coverage breadth, uniformity, and reproducibility and is superior to Repli-g for genome-wide CNV profiling and detecting focal oncogenic amplifications. For mutation analysis, none of the WGA methods were found to achieve sufficient sensitivity and specificity by whole exome sequencing. Finally, profiling of single CTCs from patients with non-small-cell lung cancer revealed potentially clinically relevant CNVs. In conclusion, MALBAC WGA coupled with low-pass whole genome sequencing is a robust workflow for genome-wide CNV profiling at single-cell level and has great potential to be applied in clinical investigations. Nevertheless, data suggest that none of the evaluated single-cell sequencing workflows can reach sufficient sensitivity or specificity for mutation detection required for clinical applications.

Carcinoma, Non-Small-Cell Lung↗

PubMed2026

On-filter fractionation by empFASP improves identification of membrane peptides in proteomic experiments.

Membrane proteins remain among the most analytically challenging targets in bottom-up proteomics due to their limited solubility and low abundance of protease-accessible sites within transmembrane domains. In addition, hydrophobic peptides are frequently lost during detergent removal and the on-filter processing steps. Here, we present empFASP, a straightforward on-filter-fractionation-based modification of the enhanced filter-aided sample preparation (eFASP) workflow that enhances recovery of membrane-embedded peptides otherwise lost during digestion and cleanup. The method combines controlled on-filter inversion with sequential ethyl acetate extraction at defined pH values, enabling recovery of peptide material retained on the filter and redistributed into detergent micelles. Compared with SP3 and SP4 in HEK293T lysates, empFASP increased unique hydrophobic peptide identifications by up to 48% and increased the proportion of detected transmembrane peptides. Application to mouse mitochondrial membranes and phosphatidylethanolamine-deficient and PE-containing Escherichia coli membranes showed that the additional fractions of empFASP contribute complementary recovery of hydrophobic and membrane-associated peptides, with the strongest gains observed at the peptide level. Because empFASP requires no specialized reagents or instrumentation, it can be readily implemented in standard proteomics workflows to improve coverage of membrane-embedded regions. SIGNIFICANCE: The empFASP (enhanced membrane peptide) workflow offers a practical solution to one of the persistent limitations in membrane proteomics-the underrepresentation of hydrophobic and transmembrane peptides in standard digests. By integrating simple pH-controlled extractions into an on-filter format, empFASP recovers peptides otherwise lost through adsorption or detergent micelle retention, substantially improving coverage of the membrane proteome. This method expands the analytical reach of bottom-up proteomics without requiring specialized instrumentation, making it immediately applicable for studies of membrane topology, protein-lipid interactions, and the structural consequences of altered membrane composition.

Proteomics↗