Whole-genome bisulfite sequencing with improved accuracy and cost.

DNA methylation patterns in the genome both reflect and help to mediate transcriptional regulatory processes. The digital nature of DNA methylation, present or absent on each allele, makes this assay capable of quantifying events in subpopulations of cells, whereas genome-wide chromatin studies lack the same quantitative capacity. Testing DNA methylation throughout the genome is possible using whole-genome bisulfite sequencing (WGBS), but the high costs associated with the assay have made it impractical for studies involving more than limited numbers of samples. We have optimized a new transposase-based library preparation assay for the Illumina HiSeq X platform suitable for limited amounts of DNA and providing a major cost reduction for WGBS. By incorporating methylated cytosines during fragment end repair, we reveal an end-repair artifact affecting 1%-2% of reads that we can remove analytically. We show that the use of a high (G + C) content spike-in performs better than PhiX in terms of bisulfite sequencing quality. As expected, the loci with transposase-accessible chromatin are DNA hypomethylated and enriched in flanking regions by post-translational modifications of histones usually associated with positive effects on gene expression. Using these transposase-accessible loci to represent the cis-regulatory loci in the genome, we compared the representation of these loci between WGBS and other genome-wide DNA methylation assays, showing WGBS to outperform substantially all of the alternatives. We conclude that it is now technologically and financially feasible to perform WGBS in larger numbers of samples with greater accuracy than previously possible.

Extracellular vesicles in DLBCL provide abundant clues to aberrant transcriptional programming and genomic alterations.

The biological role of extracellular vesicles (EVs) in diffuse large B-cell lymphoma (DLBCL) initiation and progression remains largely unknown. We characterized EVs secreted by 5 DLBCL cell lines, a primary DLBCL tumor, and a normal control B-cell sample, optimized their purification, and analyzed their content. We found that DLBCLs secreted large quantities of CD63, Alix, TSG101, and CD81 EVs, which can be extracted using an ultracentrifugation-based method and traced by their cell of origin surface markers. We also showed that tumor-derived EVs can be exchanged between lymphoma cells, normal tonsillar cells, and HK stromal cells. We then examined the content of EVs, focusing on isolation of high-quality total RNA. We sequenced the total RNA and analyzed the nature of RNA species, including coding and noncoding RNAs. We compared whole-cell and EV-derived RNA composition in benign and malignant B cells and discovered that transcripts from EVs were involved in many critical cellular functions. Finally, we performed mutational analysis and found that mutations detected in EVs exquisitely represented mutations in the cell of origin. These results enhance our understanding and enable future studies of the role that EVs may play in the pathogenesis of DLBCL, particularly with regards to the exchange of genomic information. Current findings open a new strategy for liquid biopsy approaches in disease monitoring.

The SEQC2 epigenomics quality control (EpiQC) study.

BACKGROUND: Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify types of cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and cross-validated resource for epigenetics research from the FDA's Epigenomics Quality Control Group. RESULTS: Each sample is processed in multiple replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), enzymatic deamination method (EMSeq), targeted methylation sequencing (Illumina Methyl Capture EPIC), single-molecule long-read nanopore sequencing from Oxford Nanopore Technologies, and 850k Illumina methylation arrays. After rigorous quality assessment and comparison to Illumina EPIC methylation microarrays and testing on a range of algorithms (Bismark, BitmapperBS, bwa-meth, and BitMapperBS), we find overall high concordance between assays, but also differences in efficiency of read mapping, CpG capture, coverage, and platform performance, and variable performance across 26 microarray normalization algorithms. CONCLUSIONS: The data provided herein can guide the use of these DNA reference materials in epigenomics research, as well as provide best practices for experimental design in future studies. By leveraging seven human cell lines that are designated as publicly available reference materials, these data can be used as a baseline to advance epigenomics research.

Genome-wide cell-free DNA mutational integration enables ultra-sensitive cancer monitoring.

In many areas of oncology, we lack sensitive tools to track low-burden disease. Although cell-free DNA (cfDNA) shows promise in detecting cancer mutations, we found that the combination of low tumor fraction (TF) and limited number of DNA fragments restricts low-disease-burden monitoring through the prevailing deep targeted sequencing paradigm. We reasoned that breadth may supplant depth of sequencing to overcome the barrier of cfDNA abundance. Whole-genome sequencing (WGS) of cfDNA allowed ultra-sensitive detection, capitalizing on the cumulative signal of thousands of somatic mutations observed in solid malignancies, with TF detection sensitivity as low as 10-5. The WGS approach enabled dynamic tumor burden tracking and postoperative residual disease detection, associated with adverse outcome. Thus, we present an orthogonal framework for cfDNA cancer monitoring via genome-wide mutational integration, enabling ultra-sensitive detection, overcoming the limitation of cfDNA abundance and empowering treatment optimization in low-disease-burden oncology care.

Lack of cross-species transmission of porcine endogenous retrovirus (PERV) to transplant recipients and abattoir workers in contact with pigs.

This study investigated the potential transmission of porcine endogenous retrovirus (PERV) to solid-organ transplant recipients and abattoir workers in contact with pigs. Blood samples were obtained from volunteer healthy blood donors (Group A; n=33); pig-breeding farmers who had undergone a liver transplant (Group B; n=14); and pig abattoir workers (Group C; n=49). A second blood sample was obtained 1 year after the first sample from 10 of the abattoir workers (Group D). Tests included investigation for PERV-DNA, PERV-RNA, pig-specific mitochondrial DNA, a quantitative detection of PERV nucleic acids, and antibodies to PERV by two different Western Blots. All polymerase chain reaction and Western Blots assays were negative for PERV or antibodies to PERV. Therefore, the risks of cross-species transmission of PERV appear to be negligible for immunocompetent individuals and allotransplant recipients, even if they are in close and repeated contact with live pigs or pig tissues.

Enhanced reduced representation bisulfite sequencing for assessment of DNA methylation at base pair resolution.

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

DNA Methylation Dynamics of Germinal Center B Cells Are Mediated by AID.

Changes in DNA methylation are required for the formation of germinal centers (GCs), but the mechanisms of such changes are poorly understood. Activation-induced cytidine deaminase (AID) has been recently implicated in DNA demethylation through its deaminase activity coupled with DNA repair. We investigated the epigenetic function of AID in vivo in germinal center B cells (GCBs) isolated from wild-type (WT) and AID-deficient (Aicda(-/-)) mice. We determined that the transit of B cells through the GC is associated with marked locus-specific loss of methylation and increased methylation diversity, both of which are lost in Aicda(-/-) animals. Differentially methylated cytosines (DMCs) between GCBs and naive B cells (NBs) are enriched in genes that are targeted for somatic hypermutation (SHM) by AID, and these genes form networks required for B cell development and proliferation. Finally, we observed significant conservation of AID-dependent epigenetic reprogramming between mouse and human B cells.

A MicroRNA feedback circuit in midbrain dopamine neurons.

MicroRNAs (miRNAs) are evolutionarily conserved, 18- to 25-nucleotide, non-protein coding transcripts that posttranscriptionally regulate gene expression during development. miRNAs also occur in postmitotic cells, such as neurons in the mammalian central nervous system, but their function is less well characterized. We investigated the role of miRNAs in mammalian midbrain dopaminergic neurons (DNs). We identified a miRNA, miR-133b, that is specifically expressed in midbrain DNs and is deficient in midbrain tissue from patients with Parkinson's disease. miR-133b regulates the maturation and function of midbrain DNs within a negative feedback circuit that includes the paired-like homeodomain transcription factor Pitx3. We propose a role for this feedback circuit in the fine-tuning of dopaminergic behaviors such as locomotion.

Identification of receptors for pig endogenous retrovirus.

Xenotransplantation of porcine tissues has the potential to treat a wide variety of major health problems including organ failure and diabetes. Balanced against the potential benefits of xenotransplantation, however, is the risk of human infection with a porcine microorganism. In particular, the transmission of porcine endogenous retrovirus (PERV) is a major concern [Chapman, L. E. & Bloom, E. T. (2001) J. Am. Med. Assoc. 285, 2304-2306]. Here we report the identification of two, sequence-related, human proteins that act as receptors for PERV-A, encoded by genes located on chromosomes 8 and 17. We also describe homologs from baboon and porcine cells that also are active as receptors. Conversely, activity could not be demonstrated with a syntenic murine receptor homolog. Sequence analysis indicates that PERV-A receptors [human PERV-A receptor (HuPAR)-1, HuPAR-2, baboon PERV-A receptor 2, and porcine PERV-A receptor] are multiple membrane-spanning proteins similar to receptors for other gammaretroviruses. Expression is widespread in human tissues including peripheral blood mononuclear cells, but their biological functions are unknown. The identification of the PERV-A receptors opens avenues of research necessary for a more complete assessment of the retroviral risks of pig to human xenotransplantation.