Sequencing of N6-methyl-deoxyadenosine at single-base resolution across the mammalian genome.

Although DNA N6-methyl-deoxyadenosine (6mA) is abundant in bacteria and protists, its presence and function in mammalian genomes have been less clear. We present Direct-Read 6mA sequencing (DR-6mA-seq), an antibody-independent method, to measure 6mA at base resolution. DR-6mA-seq employs a unique mutation-based strategy to reveal 6mA sites as misincorporation signatures without any chemical or enzymatic modulation of 6mA. We validated DR-6mA-seq through the successful mapping of the well-characterized G(6mA)TC motif in the E. coli DNA. As expected, when applying DR-6mA-seq to mammalian systems, we found that genomic DNA (gDNA) 6mA abundance is generally low in most mammalian tissues and cells; however, we did observe distinct gDNA 6mA sites in mouse testis and glioblastoma cells. DR-6mA-seq provides an enabling tool to detect 6mA at single-base resolution for a comprehensive understanding of DNA 6mA in eukaryotes.

5-Hydroxymethylcytosine signals in serum are a predictor of chemoresistance in high-grade serous ovarian cancer.

OBJECTIVE: The genome-wide profiling of 5-hydroxymethylcytosines (5hmC) on circulating cell-free DNA (cfDNA) has revealed promising biomarkers for various diseases. The purpose of this study was to investigate 5hmC signals in serum cfDNA and identify novel predictive biomarkers for the development of chemoresistance in high-grade serous ovarian cancer (HGSOC). We hypothesized that 5hmC profiles in cfDNA reflect the development of chemoresistance and elucidate pathways that may drive chemoresistance in HGSOC. Moreover, we sought to identify predictors that would better stratify outcomes for women with intermediate-sensitive HGSOC. METHODS: Women diagnosed with HGSOC and known platinum sensitivity status were selected for this study. Nano-hmC-Seal was performed on cfDNA isolated from archived serum samples, and differential 5hmC features were identified using DESeq2 to establish a model predictive of chemoresistance. RESULTS: A multivariate model consisting of three features (preoperative CA-125, largest residual implant after surgery, 5hmC level of OSGEPL), stratified samples from intermediate sensitive, chemo-naive women diagnosed with HGSOC into chemotherapy-resistant- and sensitive-like strata with a significant difference in overall survival (OS). Independent analysis of The Cancer Genome Atlas data further confirmed that high OSGEPL1 expression is a favorable prognostic factor for HGSOC. CONCLUSIONS: We have developed a novel multivariate model based on clinico-pathologic data and a cfDNA-derived 5hmC modified gene, OSGEPL1, that predicted response to platinum-based chemotherapy in intermediate-sensitive HGSOC. Our multivariate model applies to chemo-naïve samples regardless if the patint was treated with adjuvant or neoadjuvant chemotherapy. These results merit further investigation of the predictive capability of our model in larger cohorts.

Comparative analysis of the microbiomes of strawberry wild species Fragaria nilgerrensis and cultivated variety Akihime using amplicon-based next-generation sequencing.

Fragaria nilgerrensis is a wild strawberry species widely distributed in southwest China and has strong ecological adaptability. Akihime (F. × ananassa Duch. cv. Akihime) is one of the main cultivated strawberry varieties in China and is prone to infection with a variety of diseases. In this study, high-throughput sequencing was used to analyze and compare the soil and root microbiomes of F. nilgerrensis and Akihime. Results indicate that the wild species F. nilgerrensis showed higher microbial diversity in nonrhizosphere soil and rhizosphere soil and possessed a more complex microbial network structure compared with the cultivated variety Akihime. Genera such as Bradyrhizobium and Anaeromyxobacter, which are associated with nitrogen fixation and ammonification, and Conexibacter, which is associated with ecological toxicity resistance, exhibited higher relative abundances in the rhizosphere and nonrhizosphere soil samples of F. nilgerrensis compared with those of Akihime. Meanwhile, the ammonia-oxidizing archaea Candidatus Nitrososphaera and Candidatus Nitrocosmicus showed the opposite tendencies. We also found that the relative abundances of potential pathogenic genera and biocontrol bacteria in the Akihime samples were higher than those in the F. nilgerrensis samples. The relative abundances of Blastococcus, Nocardioides, Solirubrobacter, and Gemmatimonas, which are related to pesticide degradation, and genus Variovorax, which is associated with root growth regulation, were also significantly higher in the Akihime samples than in the F. nilgerrensis samples. Moreover, the root endophytic microbiomes of both strawberry species, especially the wild F. nilgerrensis, were mainly composed of potential biocontrol and beneficial bacteria, making them important sources for the isolation of these bacteria. This study is the first to compare the differences in nonrhizosphere and rhizosphere soils and root endogenous microorganisms between wild and cultivated strawberries. The findings have great value for the research of microbiomes, disease control, and germplasm innovation of strawberry.

Machine learning identifies cell-free DNA 5-hydroxymethylation biomarkers that detect occult colorectal cancer in PLCO Screening Trial subjects.

Background: Colorectal cancer (CRC) is a leading cause of cancer-related mortality, and CRC detection through screening improves survival rates. A promising avenue to improve patient screening compliance is the development of minimally-invasive liquid biopsy assays that target CRC biomarkers on circulating cell-free DNA (cfDNA) in peripheral plasma. In this report, we identify cfDNA biomarker candidate genes bearing the epigenetic mark 5-hydroxymethylcytosine (5hmC) that diagnose occult CRC up to 36 months prior to clinical diagnosis using the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial samples. Methods: Archived PLCO Trial plasma samples containing cfDNA were obtained from the National Cancer Institute (NCI) biorepositories. Study subjects included those who were diagnosed with CRC within 36 months of blood collection (i.e., case, n = 201) and those who were not diagnosed with any cancer during an average of 16.3 years of follow-up (i.e., controls, n = 402). Following the extraction of 3 - 8 ng cfDNA from less than 300 microliters plasma, we employed the sensitive 5hmC-Seal chemical labeling approach, followed by next-generation sequencing (NGS). We then conducted association studies and machine-learning modeling to analyze the genome-wide 5hmC profiles within training and validation groups that were randomly selected at a 2:1 ratio. Results: Despite the technical challenges associated with the PLCO samples (e.g., limited plasma volumes, low cfDNA amounts, and long archival times), robust genome-wide 5hmC profiles were successfully obtained from these samples. Association analyses using the Cox proportional hazards models suggested several epigenetic pathways relevant to CRC development distinguishing cases from controls. A weighted Cox model, comprised of 32-associated gene bodies, showed predictive detection value for CRC as early as 24-36 months prior to overt tumor presentation, and a trend for increased predictive power was observed for blood samples collected closer to CRC diagnosis. Notably, the 5hmC-based predictive model showed comparable performance regardless of sex and self-reported race/ethnicity, and significantly outperformed risk factors such as age and obesity according to BMI (body mass index). Additionally, further improvement of predictive performance was achieved by combining the 5hmC-based model and risk factors for CRC. Conclusions: An assay of 5hmC epigenetic signals on cfDNA revealed candidate biomarkers with the potential to predict CRC occurrence despite the absence of clinical symptoms or the availability of effective predictors. Developing a minimally-invasive clinical assay that detects 5hmC-modified biomarkers holds promise for improving early CRC detection and ultimately patient survival through higher compliance screening and earlier intervention. Future investigation to expand this strategy to prospectively collected samples is warranted.

A 5-Hydroxymethylcytosine-Based Noninvasive Model for Early Detection of Colorectal Carcinomas and Advanced Adenomas: The METHOD-2 Study.

PURPOSE: Detection of colorectal carcinomas at a time when there are more treatment options is associated with better outcomes. This prospective case-control study assessed the 5-hydroxymethylcytosine (5hmC) biomarkers in circulating cell-free DNA (cfDNA) for early detection of colorectal carcinoma and advanced adenomas (AA). EXPERIMENTAL DESIGN: Plasma cfDNA samples from 2,576 study participants from the multicenter METHOD-2 study (NCT03676075) were collected, comprising patients with newly diagnosed colorectal carcinoma (n = 1,074), AA (n = 356), other solid tumors (n = 80), and non-colorectal carcinoma/AA controls (n = 1,066), followed by genome-wide 5hmC profiling using the 5hmC-Seal technique and the next-generation sequencing. A weighted diagnostic model for colorectal carcinoma (stage I-III) and AA was developed using the elastic net regularization in a discovery set and validated in independent samples. RESULTS: Distribution of 5hmC in cfDNA reflected gene regulatory relevance and tissue of origin. Besides being confirmed in internal validation, a 96-gene model achieved an area under the curve (AUC) of 90.7% for distinguishing stage I-III colorectal carcinoma from controls in 321 samples from multiple centers for external validation, regardless of primary location or mutation status. This model also showed cancer-type specificity as well as high capacity for distinguishing AA from controls with an AUC of 78.6%. Functionally, differential 5hmC features associated with colorectal carcinoma and AA demonstrated relevance to colorectal carcinoma biology, including pathways such as calcium and MAPK signaling. CONCLUSIONS: Genome-wide mapping of 5hmC in cfDNA shows promise as a highly sensitive and specific noninvasive blood test to be integrated into screening programs for improving early detection of colorectal carcinoma and high-risk AA.

LABS: linear amplification-based bisulfite sequencing for ultrasensitive cancer detection from cell-free DNA.

Methylation-based liquid biopsies show promises in detecting cancer using circulating cell-free DNA; however, current limitations impede clinical application. Most assays necessitate substantial DNA inputs, posing challenges. Additionally, underrepresented tumor DNA fragments may go undetected during exponential amplification steps of traditional sequencing methods. Here, we report linear amplification-based bisulfite sequencing (LABS), enabling linear amplification of bisulfite-treated DNA fragments in a genome-wide, unbiased fashion, detecting cancer abnormalities with sub-nanogram inputs. Applying LABS to 100 patient samples revealed cancer-specific patterns, copy number alterations, and enhanced cancer detection accuracy by identifying tissue-of-origin and immune cell composition.