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Somatic mosaicism is an important cause of disease, but mosaic and somatic variants are often challenging to detect because they exist in only a fraction of cells. To address the need for benchmarking subclonal variants in normal cell populations, we developed a benchmark containing mosaic variants in the Genome in a Bottle Consortium (GIAB) HG002 reference material DNA from a large batch of a normal lymphoblastoid cell line. First, we used a somatic variant caller with high coverage (300x) Illumina whole genome sequencing data from the Ashkenazi Jewish trio to detect variants in HG002 not detected in at least 5% of cells from the combined parental data. These candidate mosaic variants were subsequently evaluated using >100x BGI, Element, and PacBio HiFi data. High confidence candidate SNVs with variant allele fractions above 5% were included in the HG002 draft mosaic variant benchmark, with 13/85 occurring in medically relevant gene regions. We also delineated a 2.45 Gbp subset of the previously defined germline autosomal benchmark regions for HG002 in which no additional mosaic variants >2% exist, enabling robust assessment of false positives. The variant allele fraction of some mosaic variants is different between batches of cells, so using data from the homogeneous batch of reference material DNA is critical for benchmarking these variants. External validation of this mosaic benchmark showed it can be used to reliably identify both false negatives and false positives for a variety of technologies and detection algorithms, demonstrating its utility for optimization and validation. By adding our characterization of mosaic variants in this widely-used cell line, we support extensive benchmarking efforts using it in simulation, spike-in, and mixture studies., Competing Interests: Competing Interests C.E.M. is a co-Founder of Onegevity. Y.W., M.R., A.V., L.M., W.C., S.C., J.H., R.M., and G.P. are Illumina employees and equity owners. A.C., P.C., K.S., D.C., A.K., and L.B. are employees of Google LLC and receive equity compensation. P.C.B. sits on the scientific advisory boards of Intersect Diagnostics Inc. and BioSymetrics Inc., and previously sat on that of Sage Bionetworks.

This proof-of-concept evaluation demonstrates that next-generation sequencing-based liquid biopsy can detect genomic alterations in the blood of cats with cancer and the absence of such alterations in the blood of presumably cancer-free cats. Two cats with cytologically confirmed lymphoma and nine presumably cancer-free cats were included in this analysis. Whole blood was collected from each subject and samples were subjected to DNA extraction, library preparation, and next-generation sequencing. Both cancer-diagnosed subjects had somatic copy number variants (a "cancer signal") identified in cell-free DNA, suggesting the current presence of cancer in these subjects. All nine presumably cancer-free subjects had unremarkable genomic profiles, suggesting the absence of cancer in these subjects. Liquid biopsy using next-generation sequencing of cell-free DNA allows for blood-based detection of cancer-associated genomic alterations in cats. Such technology has the potential to offer considerable utility in veterinary medicine, particularly for the non-invasive prioritization of small cell intestinal lymphoma versus inflammatory bowel disease in cats with gastrointestinal signs. This study lays the foundation for future studies to fully validate this type of testing for use in clinical practice., Competing Interests: CR-P, PN, MM, FM, TT, LM, MK, BF, SG, JR, DG, SH, JT, DT, AF, KK were employed by PetDx. This study received funding from PetDx. The funder had the following involvement with the study: study design, data collection and analysis, decision to publish, and preparation of the manuscript., (Copyright © 2024 Ruiz-Perez, Nakashe, Marshall, Marass, Tang, McLennan, Kroll, Flesner, Gray, Rafalko, Grosu, Hicks, Tynan, Tsui, Flory and Kruglyak.)

Objective: To validate the performance of a novel, integrated test for canine cancer screening that combines cell-free DNA quantification with next-generation sequencing (NGS) analysis., Sample: Retrospective data from a total of 1,947 cancer-diagnosed and presumably cancer-free dogs were used to validate test performance for the detection of 7 predefined cancer types (lymphoma, hemangiosarcoma, osteosarcoma, leukemia, histiocytic sarcoma, primary lung tumors, and urothelial carcinoma), using independent training and testing sets., Methods: Cell-free DNA quantification data from all samples were analyzed using a proprietary machine learning algorithm to determine a Cancer Probability Index (High, Moderate, or Low). High and Low Probability of Cancer were final result classifications. Moderate cases were additionally analyzed by NGS to arrive at a final classification of High Probability of Cancer (Cancer Signal Detected) or Low Probability of Cancer (Cancer Signal Not Detected)., Results: Of the 595 dogs in the testing set, 89% (n = 530) received a High or Low Probability result based on the machine learning algorithm; 11% (65) were Moderate Probability, and NGS results were used to assign a final classification. Overall, 87 of 122 dogs with the 7 predefined cancer types were classified as High Probability and 467 of 473 presumably cancer-free dogs were classified as Low Probability, corresponding to a sensitivity of 71.3% for the predefined cancer types at a specificity of 98.7%., Clinical Relevance: This integrated test offers a novel option to screen for cancer types that may be difficult to detect by physical examination at a dog's wellness visit.

Objective: The purpose of this study was to evaluate the performance of a next-generation sequencing-based liquid biopsy test for cancer monitoring in dogs., Samples: Pre- and postoperative blood samples were collected from dogs with confirmed cancer diagnoses originally enrolled in the CANcer Detection in Dogs (CANDiD) study. A subset of dogs also had longitudinal blood samples collected for recurrence monitoring., Methods: All cancer-diagnosed patients had a preoperative blood sample in which a cancer signal was detected and had at least 1 postoperative sample collected. Clinical data were used to assign a clinical disease status for each follow-up visit., Results: Following excisional surgery, in the absence of clinical residual disease at the postoperative visit, patients with Cancer Signal Detected results at that visit were 1.94 times as likely (95% CI, 1.21 to 3.12; P = .013) to have clinical recurrence within 6 months compared to patients with Cancer Signal Not Detected results. In the subset of patients with longitudinal liquid biopsy samples that had clinical recurrence documented during the study period, 82% (9/11; 95% CI, 48% to 97%) had Cancer Signal Detected in blood prior to or concomitant with clinical recurrence; in the 6 patients where molecular recurrence was detected prior to clinical recurrence, the median lead time was 168 days (range, 47 to 238)., Clinical Relevance: Next-generation sequencing-based liquid biopsy is a noninvasive tool that may offer utility as an adjunct to current standard-of-care clinical assessment for cancer monitoring; further studies are needed to confirm diagnostic accuracy in a larger population.

Age-related somatic genomic alterations in hematopoietic cell lines have been well characterized in humans; however, this phenomenon has not been well studied in other species. Next-generation sequencing-based liquid biopsy testing for cancer detection was recently developed for dogs and has been used to study the genomic profiles of blood samples from thousands of canine patients since 2021. In this study, 4870 client-owned dogs with and without a diagnosis or suspicion of cancer underwent liquid biopsy testing by this method. Copy number variants detected exclusively in genomic DNA derived from white blood cells (WBC gDNA-specific CNVs) were observed in 126 dogs (2.6%; 95% CI: 2.2-3.1); these copy number variants were absent from matched plasma cell-free DNA, and from tumor tissue in dogs with concurrent cancer. These findings were more common in older dogs and were persistent in WBC gDNA in over 70% of patients, with little to no change in the amplitude of the signal across longitudinal samples. Many of these alterations were observed at recurrent locations in the genome across subjects; the most common finding was a partial loss on CFA25, typically accompanied by a partial gain on the same chromosome. These early findings suggest that age-related somatic alterations may be present at an appreciable frequency in the general canine population. Further research is needed to determine the clinical significance of these findings.

Objective: To review ordering patterns, positivity rates, and outcome data for a subset of consecutive samples submitted for a commercially available, blood-based multicancer early-detection liquid biopsy test for dogs using next-generation sequencing at 1 laboratory., Sample: 1,500 consecutively submitted blood samples from client-owned dogs with and without clinical suspicion and/or history of cancer for prospective liquid biopsy testing between December 28, 2021, and June 28, 2022., Procedures: We performed a retrospective observational study, reviewing data from 1,500 consecutive clinical samples submitted for liquid biopsy testing. Outcome data were obtained via medical record review, direct communication with the referring clinic, and/or a patient outcome survey through October 16, 2022., Results: Sixty-four percent (910/1,419) of reportable samples were submitted for cancer screening, 26% (366/1,419) for aid in diagnosis, and 10% (143/1,419) for other indications. The positivity rate was 25.4% (93/366) in aid-in-diagnosis patients and 4.5% (41/910) in screening patients. Outcome data were available for 33% (465/1,401) of patients, and outcomes were classifiable for 428 patients. The relative observed sensitivity was 61.5% (67/109) and specificity was 97.5% (311/319). The positive predictive value was 75.0% (21/28) for screening patients and 97.7% (43/44) for aid-in-diagnosis patients, and the time to diagnostic resolution following a positive result was < 2 weeks in most cases., Clinical Relevance: Liquid biopsy using next-generation sequencing represents a novel tool for noninvasive detection of cancer in dogs. Real-world clinical performance meets or exceeds expectations established in the test's clinical validation study.

Cancer is the leading cause of death in dogs, yet there are no established screening paradigms for early detection. Liquid biopsy methods that interrogate cancer-derived genomic alterations in cell-free DNA in blood are being adopted for multi-cancer early detection in human medicine and are now available for veterinary use. The CANcer Detection in Dogs (CANDiD) study is an international, multi-center clinical study designed to validate the performance of a novel multi-cancer early detection "liquid biopsy" test developed for noninvasive detection and characterization of cancer in dogs using next-generation sequencing (NGS) of blood-derived DNA; study results are reported here. In total, 1,358 cancer-diagnosed and presumably cancer-free dogs were enrolled in the study, representing the range of breeds, weights, ages, and cancer types seen in routine clinical practice; 1,100 subjects met inclusion criteria for analysis and were used in the validation of the test. Overall, the liquid biopsy test demonstrated a 54.7% (95% CI: 49.3-60.0%) sensitivity and a 98.5% (95% CI: 97.0-99.3%) specificity. For three of the most aggressive canine cancers (lymphoma, hemangiosarcoma, osteosarcoma), the detection rate was 85.4% (95% CI: 78.4-90.9%); and for eight of the most common canine cancers (lymphoma, hemangiosarcoma, osteosarcoma, soft tissue sarcoma, mast cell tumor, mammary gland carcinoma, anal sac adenocarcinoma, malignant melanoma), the detection rate was 61.9% (95% CI: 55.3-68.1%). The test detected cancer signal in patients representing 30 distinct cancer types and provided a Cancer Signal Origin prediction for a subset of patients with hematological malignancies. Furthermore, the test accurately detected cancer signal in four presumably cancer-free subjects before the onset of clinical signs, further supporting the utility of liquid biopsy as an early detection test. Taken together, these findings demonstrate that NGS-based liquid biopsy can offer a novel option for noninvasive multi-cancer detection in dogs., Competing Interests: AF, KMK, JAT, LMM, JMR, PCF, GEH, FM, PN, CAR-P, DMF, TJ, RM-P, KW, ALM-W, DSG, AP, KM, SCH, KML, LH, JC, IC, and DWYT are employed by or affiliated with PetDx; and receive compensation from PetDx and/or hold vested or unvested equity in PetDx. Drs. Flesner and Leibman are members of the PetDx clinical advisory board and receive compensation and equity. Dr. Borgatti has ownership interest (including patents) in a patent entitled “Reduction of EGFR therapeutic toxicity” filed by the University of Minnesota Office of Technology Commercialization. Drs. Cahalane, Phillips, and Aiken are investors in PetDx. Drs. Jensen, van den Boom, Dharajiya, and Diaz are advisors for PetDx and hold vested or unvested equity in PetDx. Drs. Chorny, Kruglyak, Grosu, Marass, Ruiz-Perez and Tsui hold pending patent applications related to technology described in this work. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Mapping of short metagenomic (or metatranscriptomic) read data to reference isolate or single-cell genomes or metagenome-assembled genomes (MAGs) to assess microbial population relative abundance and/or structure represents an essential task of many studies across environmental and clinical settings. The filtering for the quality of the read match and assessment of read mapping results are frequently performed without visual aids or with the assistance of visualizations produced through ad-hoc, in-house approaches. Here, we introduce RecruitPlotEasy, a fully automated, user-friendly pipeline for these purposes that integrates statistical approaches to quantify intra-population sequence and gene-content diversity and identify co-occurring relative populations in the sample. Hence, RecruitPlotEasy should also greatly facilitate population genetics studies. RecruitPlotEasy is implemented in Python and R languages and is freely available open source software under the Artistic License 2.0 from https://github.com/KGerhardt/RecruitPlotEasy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gerhardt, Ruiz-Perez, Rodriguez-R, Conrad and Konstantinidis.)

The use of enterococci as a fecal indicator bacterial group for public health risk assessment has been brought into question by recent studies showing that "naturalized" populations of Enterococcus faecalis exist in the extraenteric environment. The extent to which these naturalized E. faecalis organisms can confound water quality monitoring is unclear. To determine if strains isolated from different habitats display different survival strategies and responses, we compared the decay patterns of three E. faecalis isolates from the natural environment (environmental strains) against three human gut isolates (enteric strains) in laboratory mesocosms that simulate an oligotrophic, aerobic freshwater environment. Our results showed similar overall decay rates between enteric and environmental isolates based on viable plate and quantitative PCR (qPCR) counts. However, the enteric isolates exhibited a spike in copy number ratios of 16S rRNA gene transcripts to 16S rRNA gene DNA copies (rRNA:rDNA ratios) between days 1 and 3 of the mesocosm incubations that was not observed in environmental isolates, which could indicate a different stress response. Nevertheless, there was no strong evidence of differential gene expression between environmental and enteric isolates related to habitat adaptation in the accompanying mesocosm metatranscriptomes. Overall, our results provide novel information on how rRNA levels may vary over different growth conditions (e.g., standard lab versus oligotrophic) for this important indicator bacteria. We also observed some evidence for habitat adaptation in E. faecalis; however, this adaptation may not be substantial or consistent enough for integration in water quality monitoring. IMPORTANCE Enterococci are commonly used worldwide to monitor environmental fecal contamination and public health risk for waterborne diseases. However, closely related enterococci strains adapted to living in the extraenteric environment may represent a lower public health risk and confound water quality estimates. We developed an rRNA:rDNA viability assay for E. faecalis (a predominant species within this fecal group) and tested it against both enteric and environmental isolates in freshwater mesocosms to assess whether this approach can serve as a more sensitive water quality monitoring tool. We were unable to reliably distinguish the different isolate types using this assay under the conditions tested; thus, environmental strains should continue to be counted during routine water monitoring. However, this assay could be useful for distinguishing more recent (i.e., higher-risk) fecal pollution because rRNA levels significantly decreased after 1 week in all isolates.

The diverse and ubiquitous members of the SAR11 lineage (Alphaproteobacteria) represent up to 30-40% of the surface and mesopelagic oceanic microbial communities. However, the molecular and ecological mechanisms that differentiate closely related, yet distinct, SAR11 members that often co-occur under similar environmental conditions remain speculative. Recently, two mesopelagic and oxygen minimum zone (OMZ)-associated subclades of SAR11 (Ic and IIa.A) were described using single-cell amplified genomes (SAGs) linked to nitrate reduction in OMZs. In this current study, the collection of genomes belonging to these two subclades was expanded with thirteen new metagenome-assembled genomes (MAGs), thus providing a more detailed phylogenetic and functional characterization of these subclades. Gene content-based predictions of metabolic functions revealed similarities in central carbon metabolism between subclades Ic and IIa.A and surface SAR11 clades, with small variations in central pathways. These variations included more versatile sulfur assimilation pathways, as well as a previously predicted capacity for nitrate reduction that conferred unique versatility on mesopelagic-adapted clades compared to their surface counterparts. Finally, consistent with previously reported abundances of carbon monoxide (CO) in surface and mesopelagic waters, subclades Ia (surface) and Ic (mesopelagic) have the genetic potential to oxidize carbon monoxide (CO), presumably taking advantage of this abundant compound as an electron donor. Based on genomic analyses, environmental distribution and metabolic reconstruction, we propose two new SAR11 genera, Ca. Mesopelagibacter carboxydoxydans (subclade Ic) and Ca. Anoxipelagibacter denitrificans (subclade IIa.A), which represent members of the mesopelagic and OMZ-adapted SAR11 clades., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Background: High-throughput sequencing has increased the number of available microbial genomes recovered from isolates, single cells, and metagenomes. Accordingly, fast and comprehensive functional gene annotation pipelines are needed to analyze and compare these genomes. Although several approaches exist for genome annotation, these are typically not designed for easy incorporation into analysis pipelines, do not combine results from different annotation databases or offer easy-to-use summaries of metabolic reconstructions, and typically require large amounts of computing power for high-throughput analysis not available to the average user., Results: Here, we introduce MicrobeAnnotator, a fully automated, easy-to-use pipeline for the comprehensive functional annotation of microbial genomes that combines results from several reference protein databases and returns the matching annotations together with key metadata such as the interlinked identifiers of matching reference proteins from multiple databases [KEGG Orthology (KO), Enzyme Commission (E.C.), Gene Ontology (GO), Pfam, and InterPro]. Further, the functional annotations are summarized into Kyoto Encyclopedia of Genes and Genomes (KEGG) modules as part of a graphical output (heatmap) that allows the user to quickly detect differences among (multiple) query genomes and cluster the genomes based on their metabolic similarity. MicrobeAnnotator is implemented in Python 3 and is freely available under an open-source Artistic License 2.0 from https://github.com/cruizperez/MicrobeAnnotator ., Conclusions: We demonstrated the capabilities of MicrobeAnnotator by annotating 100 Escherichia coli and 78 environmental Candidate Phyla Radiation (CPR) bacterial genomes and comparing the results to those of other popular tools. We showed that the use of multiple annotation databases allows MicrobeAnnotator to recover more annotations per genome compared to faster tools that use reduced databases and is computationally efficient for use in personal computers. The output of MicrobeAnnotator can be easily incorporated into other analysis pipelines while the results of other annotation tools can be seemingly incorporated into MicrobeAnnotator to generate summary plots.

Bacteriophages encode host-acquired functional genes known as auxiliary metabolic genes (AMGs). Photosynthesis AMGs are commonly found in marine cyanobacteria-infecting Myoviridae and Podoviridae cyanophages, but their ecology remains understudied in freshwater environments. To advance knowledge of this issue, we analysed viral metagenomes collected in the summertime for four years from five lakes and two estuarine locations interconnected by the Chattahoochee River, Southeast USA. Sequences representing ten different AMGs were recovered and found to be prevalent in all sites. Most freshwater AMGs were 10-fold less abundant than estuarine and marine AMGs and were encoded by novel Myoviridae and Podoviridae cyanophage genera. Notably, several of the corresponding viral genomes showed endemism to a specific province along the river. This translated into psbA gene phylogenetic clustering patterns that matched a marine vs. freshwater origin indicating that psbA may serve as a robust classification and source-tracking biomarker. Genomes classified in a novel viral lineage represented by isolate S-EIVl contained psbA, which is unprecedented for this lineage. Collectively, our findings indicated that the acquisition of photosynthesis AMGs is a widespread strategy used by cyanophages in aquatic ecosystems, and further indicated the existence of viral provinces in which certain viral species and/or genotypes are locally abundant., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The ubiquitous alpha-proteobacteria of the order "Candidatus Pelagibacterales" (SAR11) are highly abundant in aquatic environments, and among them, members of the monophyletic lineage LD12 (also known as SAR11 clade IIIb) are specifically found in lacustrine ecosystems. Clade IIIb bacteria are some of the most prominent members of freshwater environments, but little is known about their biology due to the lack of genome representatives. Only recently, the first non-marine isolate was cultured and described as "Candidatus Fonsibacter ubiquis". Here, we expand the collection of freshwater IIIb representatives and describe a new IIIb species of the genus "Ca. Fonsibacter". Specifically, we assembled a collection of 67 freshwater metagenomic datasets from the interconnected lakes of the Chattahoochee River basin (GA, USA) and obtained nearly complete metagenome-assembled genomes (MAGs) representing 5 distinct IIIb subclades, roughly equivalent to species based on genomic standards, including the previously described "Ca. F. ubiquis". Genomic comparisons between members of the IIIb species revealed high similarity in gene content. However, when comparing their abundance profiles in the Chattahoochee basin and various aquatic environments, differences in temporal and spatial distributions among the distinct species were observed implying niche differentiation might be underlying the coexistence of the highly functionally similar representatives. The name Ca. Fonsibacter lacus sp. nov. is proposed for the most abundant and widespread species in the Chattahoochee River basin and various freshwater ecosystems., (Copyright © 2019 Elsevier GmbH. All rights reserved.)