Characterization and mitigation of artifacts derived from NGS library preparation due to structure-specific sequences in the human genome.

BACKGROUND: Hybridization capture-based targeted next generation sequencing (NGS) is gaining importance in routine cancer clinical practice. DNA library preparation is a fundamental step to produce high-quality sequencing data. Numerous unexpected, low variant allele frequency calls were observed in libraries using sonication fragmentation and enzymatic fragmentation. In this study, we investigated the characteristics of the artifact reads induced by sonication and enzymatic fragmentation. We also developed a bioinformatic algorithm to filter these sequencing errors. RESULTS: We used pairwise comparisons of somatic single nucleotide variants (SNVs) and insertions and deletions (indels) of the same tumor DNA samples prepared using both ultrasonic and enzymatic fragmentation protocols. Our analysis revealed that the number of artifact variants was significantly greater in the samples generated using enzymatic fragmentation than using sonication. Most of the artifacts derived from the sonication-treated libraries were chimeric artifact reads containing both cis- and trans-inverted repeat sequences of the genomic DNA. In contrast, chimeric artifact reads of endonuclease-treated libraries contained palindromic sequences with mismatched bases. Based on these distinctive features, we proposed a mechanistic hypothesis model, PDSM (pairing of partial single strands derived from a similar molecule), by which these sequencing errors derive from ultrasonication and enzymatic fragmentation library preparation. We developed a bioinformatic algorithm to generate a custom mutation "blacklist" in the BED region to reduce errors in downstream analyses. CONCLUSIONS: We first proposed a mechanistic hypothesis model (PDSM) of sequencing errors caused by specific structures of inverted repeat sequences and palindromic sequences in the natural genome. This new hypothesis predicts the existence of chimeric reads that could not be explained by previous models, and provides a new direction for further improving NGS analysis accuracy. A bioinformatic algorithm, ArtifactsFinder, was developed and used to reduce the sequencing errors in libraries produced using sonication and enzymatic fragmentation.

A comprehensive performance evaluation, comparison, and integration of computational methods for detecting and estimating cross-contamination of human samples in cancer next-generation sequencing analysis.

Cross-sample contamination is one of the major issues in next-generation sequencing (NGS)-based molecular assays. This type of contamination, even at very low levels, can significantly impact the results of an analysis, especially in the detection of somatic alterations in tumor samples. Several contamination identification tools have been developed and implemented as a crucial quality-control step in the routine NGS bioinformatic pipeline. However, no study has been published to comprehensively and systematically investigate, evaluate, and compare these computational methods in the cancer NGS analysis. In this study, we comprehensively investigated nine state-of-the-art computational methods for detecting cross-sample contamination. To explore their application in cancer NGS analysis, we further compared the performance of five representative tools by qualitative and quantitative analyses using in silico and simulated experimental NGS data. The results showed that Conpair achieved the best performance for identifying contamination and predicting the level of contamination in solid tumors NGS analysis. Moreover, based on Conpair, we developed a Python script, Contamination Source Predictor (ConSPr), to identify the source of contamination. We anticipate that this comprehensive survey and the proposed tool for predicting the source of contamination will assist researchers in selecting appropriate cross-contamination detection tools in cancer NGS analysis and inspire the development of computational methods for detecting sample cross-contamination and identifying its source in the future.

Helicobacter pylori CagA-mediated ether lipid biosynthesis promotes ferroptosis susceptibility in gastric cancer.

Helicobacter pylori, particularly cytotoxin-associated gene A (CagA)-positive strains, plays a key role in the progression of gastric cancer (GC). Ferroptosis, associated with lethal lipid peroxidation, has emerged to play an important role in malignant and infectious diseases, but the role of CagA in ferroptosis in cancer cells has not been determined. Here, we report that CagA confers GC cells sensitivity to ferroptosis both in vitro and in vivo. Mechanistically, CagA promotes the synthesis of polyunsaturated ether phospholipids (PUFA-ePLs), which is mediated by increased expression of alkylglycerone phosphate synthase (AGPS) and 1-acylglycerol-3-phosphate O-acyltransferase 3 (AGPAT3), leading to susceptibility to ferroptosis. This susceptibility is mediated by activation of the MEK/ERK/SRF pathway. SRF is a crucial transcription factor that increases AGPS transcription by binding to the AGPS promoter region. Moreover, the results demonstrated that CagA-positive cells are more sensitive to apatinib than are CagA-negative cells, suggesting that detecting the H. pylori CagA status may aid patient stratification for treatment with apatinib.

Survival analysis of comprehensive treatment in Chinese patients with metastatic melanoma: A retrospective analysis.

BACKGROUND: Most of the current progression of immune checkpoint inhibitors for malignant melanoma is based on data from Caucasians in Western countries, but the benefit of Chinese patients is limited, mainly due to different pathological subtypes. The patients in western countries are mainly skin melanoma (about 90%), while the acral and mucosal types are dominant in China, accounting for 41.8% and 22.6% respectively. Acral and mucosal melanoma have lower response rates to immunotherapy and chemotherapy. OBJECTIVE: Whether immune checkpoint inhibitors can improve the survival of Chinese patients with malignant melanoma, therefore, we conducted a retrospective analysis. METHODS: We analyzed 53 patients with metastatic melanoma treated in our hospital to evaluate the efficacy and safety of PD-1 mAb in Chinese patients with metastatic melanoma, and performed univariate and multivariate analyses of prognostic factors that may affect overall survival (OS). RESULTS: In a study of 125 patients with advanced malignant melanoma, 53 patients participated, with a median follow-up of 16 months. Among these, 69.8% died, and 30.2% remained on treatment. Median progression-free survival (PFS) was 6 months, and median OS was 19 months. Patients treated with immune checkpoint inhibitors had improved outcomes, with a median PFS of 7 months and a median OS of 24 months. Patients with bone metastasis and aberrant Lactate dehydrogenase (LDH) post-treatment had worse prognoses, while immunotherapy was a protective factor. Subgroup analysis showed the benefits of immunotherapy across various patient characteristics. No unexpected toxicities were observed. CONCLUSION: The study highlights the efficacy of immune checkpoint inhibitors, particularly PD-1 mAb, in improving survival outcomes for Chinese patients with metastatic melanoma.

Preference of juvenile tiger puffer for light spectrum and tank colours based on different body size and breeding background.

As important environmental factors, the light spectra and tank colours have not received sufficient attention. Most fishes have the ability to perceive environment, distinguish colours, and exhibit preferences or aversions towards different environments, which can provide a reference for the design of their rearing environment. Tiger puffer (Takifugu rubripes) is an important mariculture species in China and East Asia, but its preference for illumination spectra and tank colours is unclear. This study focuses on the preferences of juvenile tiger puffers for different spectra and tank background colours in different rearing backgrounds and body sizes. The experiments were conducted in a preference testing device, and the behavioural videos were recorded and analysed using a motion behaviour tracking system (EthoVision XT 12). The results show that the puffers showed preference for short-wavelength lights ((i.e., cyan, green, etc.), avoidance of long-wavelength light (i.e., red) and less stay time in the full light spectrum and dark. For tank colours, the puffers showed a preference for light background colours (i.e., white), and avoidance of deep background colours (i.e., dark, red, etc.). Fish body sizes and original breeding environment could significantly affect the selective preference of juvenile puffer (P < 0.05). Large puffers preferred green tank colour than small ones, while small ones preferred grey and red. The puffers reared in green light and grey tank for 3 months preferred green light spectrum and green tank colour compared with those reared in full spectrum and grey tank, while the fish reared in full spectrum preferred grey tank colour and area without light. It was also found that the movement rate of juvenile puffers was affected by the light spectra and tank colours and was positively correlated with light wavelength (P < 0.05). Therefore, for tiger puffer breeding, short-wavelength light spectrums (cyan, green, etc.) and light-coloured tank backgrounds (white) are recommended. Long-wavelength Light-emitting diodes and dark tank colours should be avoided in breeding. This study would provide a reference basis for fish light spectra and background colour preference studies, as well as for the improvement of breeding welfare and production efficiency of juvenile tiger puffer.

Research progress on the relationship between Paneth cells-susceptibility genes, intestinal microecology and inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a disorder of the immune system and intestinal microecosystem caused by environmental factors in genetically susceptible people. Paneth cells (PCs) play a central role in IBD pathogenesis, especially in Crohn's disease development, and their morphology, number and function are regulated by susceptibility genes. In the intestine, PCs participate in the formation of the stem cell microenvironment by secreting antibacterial particles and play a role in helping maintain the intestinal microecology and intestinal mucosal homeostasis. Moreover, PC proliferation and maturation depend on symbiotic flora in the intestine. This paper describes the interactions among susceptibility genes, PCs and intestinal microecology and their effects on IBD occurrence and development.

OncoCTMiner: streamlining precision oncology trial matching via molecular profile analysis.

By establishing omics sequencing of patient tumors as a crucial element in cancer treatment, the extensive implementation of precision oncology necessitates effective and prompt execution of clinical studies for approving molecular-targeted therapies. However, the substantial volume of patient sequencing data, combined with strict clinical trial criteria, increasingly complicates the process of matching patients to precision oncology studies. To streamline enrollment in these studies, we developed OncoCTMiner, an automated pre-screening platform for molecular cancer clinical trials. Through manual tagging of eligibility criteria for 2227 oncology trials, we identified key bio-concepts such as cancer types, genes, alterations, drugs, biomarkers and therapies. Utilizing this manually annotated corpus along with open-source biomedical natural language processing tools, we trained multiple named entity recognition models specifically designed for precision oncology trials. These models analyzed 460 952 clinical trials, revealing 8.15 million precision medicine concepts, 9.32 million entity-criteria-trial triplets and a comprehensive precision oncology eligibility criteria database. Most significantly, we developed a patient-trial matching system based on cancer patients' clinical and genetic profiles, which can seamlessly integrate with the omics data analysis platform. This system expedites the pre-screening process for potentially suitable precision oncology trials, offering patients swifter access to promising treatment options. Database URL  https://oncoctminer.chosenmedinfo.com.

A unified DNA- and RNA-based NGS strategy for the analysis of multiple types of variants at the dual nucleic acid level in solid tumors.

BACKGROUND: Targeted next-generation sequencing (NGS) is a powerful and suitable approach to comprehensively identify multiple types of variants in tumors. RNA-based NGS is increasingly playing an important role in precision oncology. Both parallel and sequential DNA- and RNA-based approaches are expensive, burdensome, and have long turnaround times, which can be impractical in clinical practice. A streamlined, unified DNA- and RNA-based NGS approach is urgently needed in clinical practice. METHODS: A DNA/RNA co-hybrid capture sequencing (DRCC-Seq) approach was designed to capture pre-capture DNA and RNA libraries in a single tube and convert them into one NGS library. The performance of the DRCC-Seq approach was evaluated by a panel of reference standards and clinical samples. RESULTS: The average depth, DNA data ratio, capture ratio, and target coverage 250 (×) of the DNA panel data had a negative correlation with an increase in the proportion of RNA probes. The SNVs, indels, fusions, and MSI status were not affected by the proportion of RNA probes, but the copy numbers of the target genes were higher than expected in the standard materials, and many unexpected gene amplifications were found using D:R (1:2) and D:R (1:4) probe panels. The optimal ratio of DNA and RNA probes in the combined probe panel was 1:1 using the DRCC-Seq approach. The DRCC-Seq approach was feasible and reliable for detecting multiple types of variants in reference standards and real-world clinical samples. CONCLUSIONS: The DRCC-Seq approach is more cost-effective, with a shorter turnaround time and lower labor requirements than either parallel or sequential targeted DNA NGS and RNA NGS. It is feasible to identify multiple genetic variations at the DNA and RNA levels simultaneously in clinical practice.

Chromosome constitution and genetic relationships of Morus spp. revealed by genomic in situ hybridization.

BACKGROUND: Mulberry (Morus spp.) is an economically important woody plant, which has been used for sericulture (silk farming) for thousands of years. The genetic background of mulberry is complex due to polyploidy and frequent hybridization events. RESULTS: Comparative genomic in situ hybridization (cGISH) and self-GISH were performed to illustrate the chromosome constitution and genetic relationships of 40 mulberry accessions belonging to 12 species and three varietas in the Morus genus and containing eight different ploidy levels. We identified six homozygous cGISH signal patterns and one heterozygous cGISH signal pattern using four genomic DNA probes. Using cGISH and self-GISH data, we defined five mulberry sections (Notabilis, Nigra, Wittiorum, and Cathayana, all contained only one species; and Alba, which contained seven closely related species and three varietas, was further divided into two subsections) and proposed the genetic relationships among them. Differential cGISH signal patterns detected in section Alba allowed us to refine the genetic relationships among the closely related members of this section. CONCLUSIONS: We propose that GISH is an efficient tool to investigate the chromosome constitution and genetic relationships in mulberry. The results obtained here can be used to guide outbreeding of heterozygous perennial crops like mulberry.

Targeting of SLC25A22 boosts the immunotherapeutic response in KRAS-mutant colorectal cancer.

KRAS is an important tumor intrinsic factor driving immune suppression in colorectal cancer (CRC). In this study, we demonstrate that SLC25A22 underlies mutant KRAS-induced immune suppression in CRC. In immunocompetent male mice and humanized male mice models, SLC25A22 knockout inhibits KRAS-mutant CRC tumor growth with reduced myeloid derived suppressor cells (MDSC) but increased CD8+ T-cells, implying the reversion of mutant KRAS-driven immunosuppression. Mechanistically, we find that SLC25A22 plays a central role in promoting asparagine, which binds and activates SRC phosphorylation. Asparagine-mediated SRC promotes ERK/ETS2 signaling, which drives CXCL1 transcription. Secreted CXCL1 functions as a chemoattractant for MDSC via CXCR2, leading to an immunosuppressive microenvironment. Targeting SLC25A22 or asparagine impairs KRAS-induced MDSC infiltration in CRC. Finally, we demonstrate that the targeting of SLC25A22 in combination with anti-PD1 therapy synergizes to inhibit MDSC and activate CD8+ T cells to suppress KRAS-mutant CRC growth in vivo. We thus identify a metabolic pathway that drives immunosuppression in KRAS-mutant CRC.

Transcriptional analysis of the dimorphic fungus Umbilicaria muehlenbergii reveals the molecular mechanism of phenotypic transition.

The lichen-forming fungus Umbilicaria muehlenbergii undergoes a phenotypic transition from a yeast-like to a pseudohyphal form. However, it remains unknown if a common mechanism is involved in the phenotypic switch of U. muehlenbergii at the transcriptional level. Further, investigation of the phenotype switch molecular mechanism in U. muehlenbergii has been hindered by incomplete genomic sequencing data. Here, the phenotypic characteristics of U. muehlenbergii were investigated after cultivation on several carbon sources, revealing that oligotrophic conditions due to nutrient stress (reduced strength PDA (potato dextrose agar) media) exacerbated the pseudohyphal growth of U. muehlenbergii. Further, the addition of sorbitol, ribitol, and mannitol exacerbated the pseudohyphal growth of U. muehlenbergii regardless of PDA medium strength. Transcriptome analysis of U. muehlenbergii grown in normal and nutrient-stress conditions revealed the presence of several biological pathways with altered expression levels during nutrient stress and related to carbohydrate, protein, DNA/RNA and lipid metabolism. Further, the results demonstrated that altered biological pathways can cooperate during pseudohyphal growth, including pathways involved in the production of protectants, acquisition of other carbon sources, or adjustment of energy metabolism. Synergistic changes in the functioning of these pathways likely help U. muehlenbergii cope with dynamic stimuli. These results provide insights into the transcriptional response of U. muehlenbergii during pseudohyphal growth under oligotrophic conditions. Specifically, the transcriptomic analysis indicated that pseudohyphal growth is an adaptive mechanism of U. muehlenbergii that facilitates its use of alternative carbon sources to maintain survival.

Detecting liquid remnants of solid tumors treated with curative intent: Circulating tumor DNA as a biomarker of minimal residual disease (Review).

Circulating tumor DNA (ctDNA) has emerged as a promising biomarker of minimal residual disease (MRD) in solid tumors. There is increasing evidence to suggest that the detection of ctDNA following curative­intent treatments has high potential in anticipating future relapse in various solid tumors. Multiple liquid biopsy technical approaches and commercial platforms, including tumor­informed and tumor­agnostic ctDNA assays, have been developed for ctDNA­based MRD detection in solid tumors. Accurate ctDNA­based MRD analysis remains a critical technical challenge due to the very low concentration of ctDNA in peripheral blood samples, particularly in cancer patients following a curative­intent surgery or treatment. The present review summarizes the current key technical approaches that can be used to analyze ctDNA in the surveillance of MRD in solid tumors and provides a brief update on current commercial assays or platforms available for ctDNA­based MRD detection. The available evidence to date supporting ctDNA as a biomarker for detection of MRD in various types of solid tumors is also reviewed. In addition, technical and biological variables and considerations in pre­analytical and analytical steps associated with ctDNA­based MRD detection are discussed.

Extraction optimization and identification of four advanced glycation-end products inhibitors from lotus leaves and interaction mechanism analysis.

Previous research indicated lotus leaves extract could effectively inhibit advanced glycation end-products (AGEs) formation, but the optimal extraction condition, bio-active compounds and interaction mechanism remain unclear. The current study was designed to optimize the extraction parameters of AGEs inhibitors from lotus leaves by bio-activity-guided approach. The bio-active compounds were enriched and identified, the interaction mechanisms of inhibitors with ovalbumin (OVA) were investigated by fluorescence spectroscopy and molecular docking. The optimum extraction parameters were solid-liquid ratio of 1:30, ethanol concentration of 70 %, ultrasonic time of 40 min, temperature of 50 °C, and power of 400 W. Isoquercitrin, hyperoside, astragalin, and trifolin were identified from the 80 % ethanol fraction of lotus leaves (80HY). Hyperoside and isoquercitrin were dominant AGEs inhibitors and accounted for 55.97 % of 80HY. Isoquercitrin, hyperoside, trifolin interacted with OVA via the same mechanism, hyperoside exhibited the strongest affinity, trifolin caused the most conformational changes.

Up-regulation of oxytocin receptors on peripheral sensory neurons mediates analgesia in chemotherapy-induced neuropathic pain.

BACKGROUND AND PURPOSE: Chemotherapy-induced neuropathic pain (CINP) currently has limited effective treatment. Although the roles of oxytocin (OXT) and the oxytocin receptor (OXTR) in central analgesia have been well documented, the expression and function of OXTR in the peripheral nervous system remain unclear. Here, we evaluated the peripheral antinociceptive profiles of OXTR in CINP. EXPERIMENTAL APPROACH: Paclitaxel (PTX) was used to establish CINP. Quantitative real-time polymerase chain reaction (qRT-PCR), in situ hybridization, and immunohistochemistry were used to observe OXTR expression in dorsal root ganglia (DRG). The antinociceptive effects of OXT were assessed by hot-plate and von Frey tests. Whole-cell patch clamp was performed to record sodium currents, excitability of DRG neurons, and excitatory synapse transmission. KEY RESULTS: Expression of OXTR in DRG neurons was enhanced significantly after PTX treatment. Activation of OXTR exhibited antinociceptive effects, by decreasing the hyperexcitability of DRG neurons in PTX-treated mice. Additionally, OXTR activation up-regulated the phosphorylation of protein kinase C (pPKC) and, in turn, impaired voltage-gated sodium currents, particularly the voltage-gated sodium channel 1.7 (NaV 1.7) current, that plays an indispensable role in PTX-induced neuropathic pain. OXT suppressed excitatory transmission in the spinal dorsal horn as well as excitatory inputs from primary afferents in PTX-treated mice. CONCLUSION AND IMPLICATIONS: The OXTR in small-sized DRG neurons is up-regulated in CINP and its activation relieved CINP by inhibiting the neural excitability by impairment of NaV 1.7 currents via pPKC. Our results suggest that OXTR on peripheral sensory neurons is a potential therapeutic target to relieve CINP.

OncoPubMiner: a platform for mining oncology publications.

Updated and expert-quality knowledge bases are fundamental to biomedical research. A knowledge base established with human participation and subject to multiple inspections is needed to support clinical decision making, especially in the growing field of precision oncology. The number of original publications in this field has risen dramatically with the advances in technology and the evolution of in-depth research. Consequently, the issue of how to gather and mine these articles accurately and efficiently now requires close consideration. In this study, we present OncoPubMiner (https://oncopubminer.chosenmedinfo.com), a free and powerful system that combines text mining, data structure customisation, publication search with online reading and project-centred and team-based data collection to form a one-stop 'keyword in-knowledge out' oncology publication mining platform. The platform was constructed by integrating all open-access abstracts from PubMed and full-text articles from PubMed Central, and it is updated daily. OncoPubMiner makes obtaining precision oncology knowledge from scientific articles straightforward and will assist researchers in efficiently developing structured knowledge base systems and bring us closer to achieving precision oncology goals.

The cholesterol uptake regulator PCSK9 promotes and is a therapeutic target in APC/KRAS-mutant colorectal cancer.

Therapeutic targeting of KRAS-mutant colorectal cancer (CRC) is an unmet need. Here, we show that Proprotein Convertase Subtilisin/Kexin type 9 (PSCK9) promotes APC/KRAS-mutant CRC and is a therapeutic target. Using CRC patient cohorts, isogenic cell lines and transgenic mice, we identify that de novo cholesterol biosynthesis is induced in APC/KRAS mutant CRC, accompanied by increased geranylgeranyl diphosphate (GGPP)─a metabolite necessary for KRAS activation. PCSK9 is the top up-regulated cholesterol-related gene. PCSK9 depletion represses APC/KRAS-mutant CRC cell growth in vitro and in vivo, whereas PCSK9 overexpression induces oncogenesis. Mechanistically, PCSK9 reduces cholesterol uptake but induces cholesterol de novo biosynthesis and GGPP accumulation. GGPP is a pivotal metabolite downstream of PCSK9 by activating KRAS/MEK/ERK signaling. PCSK9 inhibitors suppress growth of APC/KRAS-mutant CRC cells, organoids and xenografts, especially in combination with simvastatin. PCSK9 overexpression predicts poor survival of APC/KRAS-mutant CRC patients. Together, cholesterol homeostasis regulator PCSK9 promotes APC/KRAS-mutant CRC via GGPP-KRAS/MEK/ERK axis and is a therapeutic target.

METTL3 Inhibits Antitumor Immunity by Targeting m6A-BHLHE41-CXCL1/CXCR2 Axis to Promote Colorectal Cancer.

BACKGROUND & AIMS: N6-Methyladenosine (m6A) is the most prevalent RNA modification and recognized as an important epitranscriptomic mechanism in colorectal cancer (CRC). We aimed to exploit whether and how tumor-intrinsic m6A modification driven by methyltransferase like 3 (METTL3) can dictate the immune landscape of CRC. METHODS: Mettl3 knockout mice, CD34+ humanized mice, and different syngeneic mice models were used. Immune cell composition and cytokine level were analyzed by flow cytometry and Cytokine 23-Plex immunoassay, respectively. M6A sequencing and RNA sequencing were performed to identify downstream targets and pathways of METTL3. Human CRC specimens (n = 176) were used to evaluate correlation between METTL3 expression and myeloid-derived suppressor cell (MDSC) infiltration. RESULTS: We demonstrated that silencing of METTL3 in CRC cells reduced MDSC accumulation to sustain activation and proliferation of CD4+ and CD8+ T cells, and eventually suppressed CRC in ApcMin/+Mettl3+/- mice, CD34+ humanized mice, and syngeneic mice models. Mechanistically, METTL3 activated the m6A-BHLHE41-CXCL1 axis by analysis of m6A sequencing, RNA sequencing, and cytokine arrays. METTL3 promoted BHLHE41 expression in an m6A-dependent manner, which subsequently induced CXCL1 transcription to enhance MDSC migration in vitro. However, the effect was negligible on BHLHE41 depletion, CXCL1 protein or CXCR2 inhibitor SB265610 administration, inferring that METTL3 promotes MDSC migration via BHLHE41-CXCL1/CXCR2. Consistently, depletion of MDSCs by anti-Gr1 antibody or SB265610 blocked the tumor-promoting effect of METTL3 in vivo. Importantly, targeting METTL3 by METTL3-single guide RNA or specific inhibitor potentiated the effect of anti-programmed cell death protein 1 (anti-PD1) treatment. CONCLUSIONS: Our study identifies METTL3 as a potential therapeutic target for CRC immunotherapy whose inhibition reverses immune suppression through the m6A-BHLHE41-CXCL1 axis. METTL3 inhibition plus anti-PD1 treatment shows promising antitumor efficacy against CRC.

Uncovering 1058 Novel Human Enteric DNA Viruses Through Deep Long-Read Third-Generation Sequencing and Their Clinical Impact.

BACKGROUND & AIMS: Lack of viral reference genomes poses a challenge to virome study. We investigated human gut virome and its clinical implication by ultra-deep metagenomic sequencing. METHODS: We extracted sufficient viral DNA from human feces for ultra-deep PacBio sequencing (>10 µg) and Illumina sequencing (>1 µg). Upon de novo assembly and 6 stages of strict filtering, viral genomes were generated and validated in 3 cohorts of 2819 published fecal metagenomes. Diagnostic performance of assembled viruses for colorectal cancer were tested in a training cohort and 2 independent validation cohorts. Virus mapping ratio, evolutionary history, and virus status (lytic or temperate) were also examined. RESULTS: The mean amount of extracted viral DNA increased by 14-fold compared with previous protocols. We obtained PacBio long reads and Illumina short reads with 290-fold higher depth than previous studies. We assembled and validated 1178 contigs as complete viral genomes, of which 1058 were newly identified. Thirteen viral genomes (398-839 kb) that are longer than the largest bacteriophage found in humans (393 kb) were discovered. Phylogenetic tree was constructed based on Hidden Markov Models alignment scores of 4 conserved viral proteins. Incorporating our assembled genomes into the National Center for Biotechnology Information database improved the mapping ratio of published metagenomes ≤18 times. Lytic viruses (75.9% ± 12.2% of total) were predominantly present in our sample. A biomarker panel of 14 novel viruses could discriminate patients with colorectal cancer from controls with an area under the receiver operating characteristics curve of 0.87 in the training cohort, which was validated with areas under the receiver operating characteristics curve of 0.85 and 0.73 in 2 independent cohorts. CONCLUSIONS: We uncovered 1058 novel human gut viruses. These findings can contribute to clinical diagnosis, current viral reference genome, and future virome investigation.

Artificial Light Pollution with Different Wavelengths at Night Interferes with Development, Reproduction, and Antipredator Defenses of Daphnia magna.

Artificial light at night (ALAN) dramatically alters the natural daily cycle of light and may cause a suite of physiological and behavioral responses of freshwater crustacean Daphnia. Here, we conducted a life table experiment to investigate the effect of different wavelengths [white (L-L, 400-800 nm), red (R, 614 nm), green (G, 527 nm), and blue (B, 447 nm)] and flashing modes [three colors flash alternately (3-Flash), seven colors strobe alternately (7-Strobe)] of ALAN on the development, reproduction, and predator-induced defenses of Daphnia magna. The results showed that G inhibited the development of the body and tail spine of Daphnia at sexual maturity and decreased their reproduction. The 7-Strobe promoted Daphnia to develop a larger eye. There was an interaction between fish kairomones and ALAN, which intensified over time. ALAN enhanced the response of the relative tail spine length to fish kairomone at sexual maturity. In addition, L-L and R inhibited the body length of 10th instar D. magna in response to fish kairomone. Through the hierarchical cluster method, the 3-Flash and B are recommended as friendly artificial lighting to Daphnia. Our results highlight the interference effects of ALAN on Daphnia, which are helpful to assess the potential impact of light pollution on zooplankton.