Whole-genome Profiling Research Articles

Monitoring Chromatin Regulation in Planarians Using Chromatin Immunoprecipitation Followed by Sequencing (ChIP-seq).

Planarians are an accessible model system to study animal regeneration and stem cells. Over the last two decades, new molecular techniques have provided us with powerful tools to understand whole-body regeneration and pluripotent adult stem cells specifically. We describe a method for performing Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) on planarian cells that relies on FACS to isolate different cell populations followed by immunoprecipitation and library preparation for next-generation sequencing. Whole-genome profiling of histone modifications enables a greater understanding of epigenetic mechanisms in development, pluripotency, and differentiation. This protocol adds to the growing list of functional genomic approaches to study whole-body regeneration in animals.

Low-frequency somatic copy number alterations in normal human lymphocytes revealed by large-scale single-cell whole-genome profiling

Genomic-scale somatic copy number alterations in healthy humans are difficult to investigate because of low occurrence rates and the structural variations’ stochastic natures. Using a Tn5-transposase-assisted single-cell whole-genome sequencing method, we sequenced over 20,000 single lymphocytes from 16 individuals. Then, with the scale increased to a few thousand single cells per individual, we found that about 7.5% of the cells had large-size copy number alterations. Trisomy 21 was the most prevalent aneuploid event among all autosomal copy number alterations, whereas monosomy X occurred most frequently in over-30-yr-old females. In the monosomy X single cells from individuals with phased genomes and identified X-inactivation ratios in bulk, the inactive X Chromosomes were lost more often than the active ones.

Identification of DNA methylation biomarkers with potential to predict response to neoadjuvant chemotherapy in triple-negative breast cancer

Neoadjuvant chemotherapy (NAC) is used to treat triple-negative breast cancer (TNBC) prior to resection. Biomarkers that accurately predict a patient’s response to NAC are needed to individualise therapy and avoid chemotoxicity from unnecessary chemotherapy. We performed whole-genome DNA methylation profiling on diagnostic TNBC biopsy samples from the Sequential Evaluation of Tumours Undergoing Preoperative (SETUP) NAC study. We found 9 significantly differentially methylated regions (DMRs) at diagnosis which were associated with response to NAC. We show that 4 of these DMRs are associated with TNBC overall survival (P < 0.05). Our results highlight the potential of DNA methylation biomarkers for predicting NAC response in TNBC.

P.0050 Whole-genome DNA methylation profiles in Anorexia Nervosa: identification of potential epigenetic biomarkers of diagnostic and prognosis

P.0050 Whole-genome DNA methylation profiles in Anorexia Nervosa: identification of potential epigenetic biomarkers of diagnostic and prognosis

Transcriptional activity and epigenetic regulation of transposable elements in the symbiotic fungus Rhizophagus irregularis.

Arbuscular mycorrhizal (AM) fungi form mutualistic relationships with most land plant species. AM fungi have long been considered as ancient asexuals. Long-term clonal evolution would be remarkable for a eukaryotic lineage and suggests the importance of alternative mechanisms to promote genetic variability facilitating adaptation. Here, we assessed the potential of transposable elements for generating such genomic diversity. The dynamic expression of TEs during Rhizophagus irregularis spore development suggests ongoing TE activity. We find Mutator-like elements located near genes belonging to highly expanded gene families. Whole-genome epigenomic profiling of R. irregularis provides direct evidence of DNA methylation and small RNA production occurring at TE loci. Our results support a model in which TE activity shapes the genome, while DNA methylation and small RNA–mediated silencing keep their overproliferation in check. We propose that a well-controlled TE activity directly contributes to genome evolution in AM fungi.

CD19-CAR T Cells Develop Exhaustion Epigenetic Programs during a Clinical Response

Background:CD19-CAR T-cell therapy has emerged as a curative approach for patients with relapsed/refractory B-cell malignancies, including lymphoma and acute lymphoblastic leukemia (ALL). However, many patients develop recurrent disease after initial responses. Therapeutic failure is most likely due to T cell extrinsic and intrinsic mechanisms. While CD19-negative relapse has emerged as a major extrinsic mechanism, T cell intrinsic mechanisms remain elusive. T-cell exhaustion is driven by epigenetic programs, however epigenetic changes in CAR T cells pre and post infusion have not been determined longitudinally. Thus, the goal of this study was to determine the epigenetic landscape of CD19-CAR T cells pre and post infusion as an initial step to elucidate intrinsic mechanisms that limit CAR T-cell effector functions in humans.Methods/Results:A longitudinal analysis of CD8+ CD19-CAR T cell epigenetic changes was performed by whole-genome DNA methylation profiling of CAR T cells during manufacturing and from peripheral blood mononuclear cells (PBMCs) of infused patients. DNA methylation profiling was performed on samples from 15 patients enrolled on our institutional, autologous CD19-CAR T cell therapy study (NCT03573700). CAR T cell expansion and persistence was determined by measuring vector copy numbers in the PBMCs of treated patients. B cell aplasia was tracked by monitoring B cell reconstitution as an additional measure of CD19-CAR T cell function. We had previously established novel exhaustion DNA methylation datasets that delineate between progenitor and fully exhausted T cells. These datasets served as a guide for stratifying our post-infusion CAR T cells along the exhaustion developmental trajectory. Lastly, publicly available single-cell transcriptional profiles of CD19-CAR T cells from patients were mined to validate expression of the transitory exhaustion signature.Our data show that CD19-CAR T cells lose repressive DNA methylation at effector loci (e.g. PRF1, TBET) while gaining methylation at genes (e.g. LEF1, TCF7) associated with memory potential. We confirmed that these epigenetic changes are coupled to endogenous human T cell effector and memory differentiation by cross-referencing our epigenetic data with publicly available transcriptional profiles for antigen-specific effector and long-lived memory CD8 T cells from individuals vaccinated for yellow fever. Furthermore, we show that CAR T cells were unable to mount an in vivo recall response after recovery of antigen-positive B cells or disease relapse. This observation, coupled to the fact that these patients' CAR T cells developed exhaustion-associated DNA methylation programs, further supports the broader conclusion that CAR T cells acquire stable epigenetic exhaustion programs that limit their protective capacity.Conclusion:Our data demonstrate for the first time that CAR T cell expansion during a clinical response is coupled to progressive restriction of gene regulatory programs that control T cell fate potential. Furthermore, we show that CD19-CAR T cells undergo exhaustion epigenetic programs that are coupled to an inability to mount a recall response in the presence of antigen-positive normal and malignant B cells. This work lays the foundation for future efforts aimed at improving the efficacy of cellular therapy by reversing epigenetic programs that are coupled to CAR T-cell exhaustion. DisclosuresTriplett: Miltenyi: Other: Travel, meeting registration. Gottschalk: Other: Other: patents and patent applications in the field of cancer cell and gene therapy ; Catamaran Bio: Consultancy; Novartis: Consultancy; Tidal: Consultancy; Immatics: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Consultancy. Youngblood: ElevateBio: Consultancy; ElevateBio: Honoraria; AstraZeneca: Honoraria; Cell Signaling: Honoraria; Merck: Research Funding; other: Other: patents and patent applications in the field of cancer cell and gene therapy .

NyuWa Genome resource: A deep whole-genome sequencing-based variation profile and reference panel for the Chinese population.

The lack of haplotype reference panels and whole-genome sequencing resources specific to the Chinese population has greatly hindered genetic studies in the world's largest population. Here, we present the NyuWa genome resource, based on deep (26.2×) sequencing of 2,999 Chinese individuals, and construct a NyuWa reference panel of 5,804 haplotypes and 19.3 million variants, which is a high-quality publicly available Chinese population-specific reference panel with thousands of samples. Compared with other panels, the NyuWa reference panel reduces the Han Chinese imputation error rate by a margin ranging from 30% to 51%. Population structure and imputation simulation tests support the applicability of one integrated reference panel for northern and southern Chinese. In addition, a total of 22,504 loss-of-function variants in coding and noncoding genes are identified, including 11,493 novel variants. These results highlight the value of the NyuWa genome resource in facilitating genetic research in Chinese and Asian populations.

Exploring Semi-Quantitative Metagenomic Studies Using Oxford Nanopore Sequencing: A Computational and Experimental Protocol.

The gut microbiome plays a major role in chronic diseases, of which several are characterized by an altered composition and diversity of bacterial communities. Large-scale sequencing projects allowed for characterizing the perturbations of these communities. However, translating these discoveries into clinical applications remains a challenge. To facilitate routine implementation of microbiome profiling in clinical settings, portable, real-time, and low-cost sequencing technologies are needed. Here, we propose a computational and experimental protocol for whole-genome semi-quantitative metagenomic studies of human gut microbiome with Oxford Nanopore sequencing technology (ONT) that could be applied to other microbial ecosystems. We developed a bioinformatics protocol to analyze ONT sequences taxonomically and functionally and optimized preanalytic protocols, including stool collection and DNA extraction methods to maximize read length. This is a critical parameter for the sequence alignment and classification. Our protocol was evaluated using simulations of metagenomic communities, which reflect naturally occurring compositional variations. Next, we validated both protocols using stool samples from a bariatric surgery cohort, sequenced with ONT, Illumina, and SOLiD technologies. Results revealed similar diversity and microbial composition profiles. This protocol can be implemented in a clinical or research setting, bringing rapid personalized whole-genome profiling of target microbiome species.

Whole-Genome Profiles of Malay Colorectal Cancer Patients with Intact MMR Proteins.

Background: This study aimed to identify new genes associated with CRC in patients with normal mismatch repair (MMR) protein expression. Method: Whole-genome sequencing (WGS) was performed in seven early-age-onset Malay CRC patients. Potential germline genetic variants, including single-nucleotide variations and insertions and deletions (indels), were prioritized using functional and predictive algorithms. Results: An average of 3.2 million single-nucleotide variations (SNVs) and over 800 indels were identified. Three potential candidate variants in three genes—IFNE, PTCH2 and SEMA3D—which were predicted to affect protein function, were identified in three Malay CRC patients. In addition, 19 candidate genes—ANKDD1B, CENPM, CLDN5, MAGEB16, MAP3K14, MOB3C, MS4A12, MUC19, OR2L8, OR51Q1, OR51AR1, PDE4DIP, PKD1L3, PRIM2, PRM3, SEC22B, TPTE, USP29 and ZNF117—harbouring nonsense variants were prioritised. These genes are suggested to play a role in cancer predisposition and to be associated with cancer risk. Pathway enrichment analysis indicated significant enrichment in the olfactory signalling pathway. Conclusion: This study provides a new spectrum of insights into the potential genes, variants and pathways associated with CRC in Malay patients.

The whole-genome and expression profile analysis of WRKY and RGAs in Dactylis glomerata showed that DG6C02319.1 and DgWRKYs may cooperate in the immunity against rust.

Orchardgrass (Dactylis glomerata) is one of the top four perennial forages worldwide and, despite its large economic advantages, often threatened by various environmental stresses. WRKY transcription factors (TFs) can regulate a variety of plant processes, widely participate in plant responses to biotic and abiotic stresses, and are one of the largest gene families in plants. WRKYs can usually bind W-box elements specifically. In this study, we identified a total of 93 DgWRKY genes and 281 RGAs, including 65, 169 and 47 nucleotide-binding site-leucine-rich repeats (NBS-LRRs), leucine-rich repeats receptor-like protein kinases (LRR-RLKs), and leucine-rich repeats receptor-like proteins (LRR-RLPs), respectively. Through analyzing the expression of DgWRKY genes in orchardgrass under different environmental stresses, it was found that many DgWRKY genes were differentially expressed under heat, drought, submergence, and rust stress. In particular, it was found that the greatest number of genes were differentially expressed under rust infection. Consistently, GO and KEGG enrichment analysis of all genes showed that 78 DgWRKY TFs were identified in the plant–pathogen interaction pathway, with 59 of them differentially expressed. Through cis-acting element prediction, 154 RGAs were found to contain W-box elements. Among them, DG6C02319.1 (a member of the LRR-RLK family) was identified as likely to interact with 14 DGWRKYs. Moreover, their expression levels in susceptible plants after rust inoculation were first up-regulated and then down-regulated, while those in the resistant plants were always up-regulated. In general, DgWRKYs responded to both biotic stress and abiotic stress. DgWRKYs and RGAs may synergistically respond to the response of orchardgrass to rust. This study provides meaningful insight into the molecular mechanisms of WRKY proteins in orchardgrass.

Whole-genome profiling of primary cutaneous anaplastic large cell lymphoma.

Primary cutaneous anaplastic large cell lymphoma (pcALCL), a hematological neoplasm caused by skin-homing CD30+ malignant T cells, is part of the spectrum of primary cutaneous CD30+ lymphoproliferative disorders. To date, only a small number of molecular alterations have been described in pcALCL and, so far, no clear unifying theme that could explain the pathogenetic origin of the disease has emerged among patients. In order to clarify the pathogenetic basis of pcALCL, we performed high-resolution genetic profiling (genome/transcriptome) of this lymphoma (n=12) by using whole-genome sequencing, whole-exome sequencing and RNA sequencing. Our study, which uncovered novel genomic rearrangements, copy number alterations and small-scale mutations underlying this malignancy, revealed that the cell cycle, T-cell physiology regulation, transcription and signaling via the PI-3-K, MAPK and G-protein pathways are cellular processes commonly impacted by molecular alterations in patients with pcALCL. Recurrent events affecting cancer-associated genes included deletion of PRDM1 and TNFRSF14, gain of EZH2 and TNFRSF8, small-scale mutations in LRP1B, PDPK1 and PIK3R1 and rearrangements involving GPS2, LINC-PINT and TNK1. Consistent with the genomic data, transcriptome analysis uncovered upregulation of signal transduction routes associated with the PI-3-K, MAPK and G-protein pathways (e.g., ERK, phospholipase C, AKT). Our molecular findings suggest that inhibition of proliferation-promoting pathways altered in pcALCL (particularly PI-3-K/AKT signaling) should be explored as potential alternative therapy for patients with this lymphoma, especially, for cases that do not respond to first-line skin-directed therapies or with extracutaneous disease.

Whole-Genome SNP Analysis Identifies Putative Mycobacterium bovis Transmission Clusters in Livestock and Wildlife in Catalonia, Spain.

The high-resolution WGS analyses of MTBC strains have provided useful insight for determining sources of infection for animal tuberculosis. In Spain, tuberculosis in livestock is caused by Mycobacterium bovis and Mycobacterium caprae, where wildlife reservoirs play an important role. We analyzed a set of 125 M. bovis isolates obtained from livestock and wildlife from Catalonia to investigate strain diversity and identify possible sources and/or causes of infection. Whole-genome SNP profiles were used for phylogenetic reconstruction and pairwise SNP distance analysis. Additionally, SNPs were investigated to identify virulence and antimicrobial resistance factors to investigate clade-specific associations. Putative transmission clusters (≤12 SNPs) were identified, and associated epidemiological metadata were used to determine possible explanatory factors for transmission. M. bovis distribution was heterogeneous, with 7 major clades and 21 putative transmission clusters. In order of importance, the explanatory factors associated were proximity and neighborhood, residual infection, livestock-wildlife interaction, shared pasture, and movement. Genes related to lipid transport and metabolism showed the highest number of SNPs. All isolates were pyrazinamide resistant, and five were additionally resistant to isoniazid, but no clade-specific associations could be determined. Our findings highlight the importance of high-resolution molecular surveillance to monitor bovine tuberculosis dynamics in a low-prevalence setting.

Genetic Diversity and Population Structure of Medemia argun (Mart.) Wurttenb. ex H.Wendl. Based on Genome-Wide Markers

Medemia argun is a wild, dioecious palm, adapted to the harsh arid environment of the Nubian Desert in Sudan and southern Egypt. There is a concern about its conservation status, since little is known about its distribution, abundance, and genetic variation. M. argun grows on the floodplains of seasonal rivers (wadis). The continuing loss of suitable habitats in the Nubian Desert is threatening the survival of this species. We analyzed the genetic diversity, population genetic structure, and occurrence of M. argun populations to foster the development of conservation strategies for M. argun. Genotyping-by-sequencing (GBS) analyses were performed using a whole-genome profiling service. We found an overall low genetic diversity and moderate genetic structuring based on 40 single-nucleotide polymorphisms (SNPs) and 9,866 SilicoDArT markers. The expected heterozygosity of the total population (HT) equaled 0.036 and 0.127, and genetic differentiation among populations/groups (FST) was 0.052 and 0.092, based on SNP and SilicoDArT markers, respectively. Bayesian clustering analyses defined five genetic clusters that did not display any ancestral gene flow among each other. Based on SilicoDArT markers, the results of the analysis of molecular variance (AMOVA) confirmed the previously observed genetic differentiation among generation groups (23%; p &amp;lt; 0.01). Pairwise FST values indicated a genetic gap between old and young individuals. The observed low genetic diversity and its loss among generation groups, even under the detected high gene flow, show genetically vulnerable M. argun populations in the Nubian Desert in Sudan. To enrich and maintain genetic variability in these populations, conservation plans are required, including collection of seed material from genetically diverse populations and development of ex situ gene banks.

Bioorthogonal Chemical Signature Enabling Amplified Visualization of Cellular Oxidative Thymines.

Cellular oxidative thymines, 5-hydroxymethyluracil (5hmU) and 5-formyluracil (5fU), are found in the genomes of a diverse range of organisms, the distribution of which profoundly influence biological processes and living systems. However, the distribution of cellular oxidative thymines has not been explored because of lacking both specific bioorthogonal labeling and sensitivity methods for single-cell analysis. Herein, we report a bioorthogonal chemical signature enabling amplified visualization of cellular oxidative thymines in single cells. The synthesized ATP-γ-alkyne, an ATP analogue with bioorthogonal tag modified on γ-phosphate can be specifically linked to cellular 5hmU by chemoenzymatic labeling. DNA with 5-alkynephosphomethyluracil were then clicked with azide (N3)-modified 5hmU-primer. Identification of 5fU is based on selective reduction from 5fU to 5hmU, subsequent chemoenzymatic labeling of the newly generated 5hmU, and cross-linking with N3-modified 5fU-primer via click chemistry. Then, all of the 5hmU and 5fU sites are encoded with respective circularized barcodes. These barcodes are simultaneously amplified for multiplexed single-molecule imaging. The above two kinds of barcodes can be simultaneously amplified for differentiated visualization of 5hmU and 5fU in single cells. We find these two kinds of cellular oxidative thymines are spatially organized in a cell-type-dependent style with cell-to-cell heterogeneity. We also investigate their multilevel subcellular information and explore their dynamic changes during cell cycles. Further, using DNA sequencing instead of fluorescence imaging, our proposed bioorthogonal chemical signature holds great potential to offer the sequence information of these oxidative thymines in cells and may provide a reliable chemical biology approach for studying the whole-genome oxidative thymines profiles and insights into their functional role and dynamics in biology.

IRF-8/miR-451a regulates M-MDSC differentiation via the AMPK/mTOR signal pathway during lupus development

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease. Myeloid-derived suppressor cells (MDSCs) have been found to be involved in the regulation of SLE development. However, little is known about the association between MDSC subsets and the factors that draw MDSCs into abnormal expansion. This study found that the percentage of M-MDSCs increased in mice with pristane-induced lupus. Toll-like receptor (TLR)7 signal activation and high interferon-α (IFN-α) level promoted M-MDSC differentiation in vitro. Moreover, both AMP-activated protein kinase (AMPK) agonist metformin and two mammalian targets of rapamycin (mTOR) inhibitors (INK128 and rapamycin) inhibited the percentage of M-MDSCs in lupus mice as well as in the TLR7- and IFN-α-induced bone marrow (BM) differentiation into MDSCs in vitro. In terms of mechanism, whole-genome transcriptome profiling was performed by RNA sequencing, revealing that the expression of the transcription factor IRF-8 was higher in M-MDSCs isolated from pristane-induced lupus mice, compared with control mice. IRF-8 was identified to be crucial for TLR7- and IFN-α-induced BM differentiation into MDSCs in vitro. Furthermore, interferon (IFN) regulatory factor8 (IRF-8) was targeted by miR-451a in M-MDSC differentiation. Of note, metformin-modified M-MDSCs could relieve lupus symptoms in pristane-induced lupus mice. The findings revealed a novel mechanism linking IRF-8/miR-451a to M-MDSC differentiation via the AMPK/mTOR signal pathway during lupus development. This study might provide an important reference for SLE therapy by targeting M-MDSCs.

Whole-genome profiling of nasopharyngeal carcinoma reveals viral-host co-operation in inflammatory NF-κB activation and immune escape

Interplay between EBV infection and acquired genetic alterations during nasopharyngeal carcinoma (NPC) development remains vague. Here we report a comprehensive genomic analysis of 70 NPCs, combining whole-genome sequencing (WGS) of microdissected tumor cells with EBV oncogene expression to reveal multiple aspects of cellular-viral co-operation in tumorigenesis. Genomic aberrations along with EBV-encoded LMP1 expression underpin constitutive NF-κB activation in 90% of NPCs. A similar spectrum of somatic aberrations and viral gene expression undermine innate immunity in 79% of cases and adaptive immunity in 47% of cases; mechanisms by which NPC may evade immune surveillance despite its pro-inflammatory phenotype. Additionally, genomic changes impairing TGFBR2 promote oncogenesis and stabilize EBV infection in tumor cells. Fine-mapping of CDKN2A/CDKN2B deletion breakpoints reveals homozygous MTAP deletions in 32-34% of NPCs that confer marked sensitivity to MAT2A inhibition. Our work concludes that NPC is a homogeneously NF-κB-driven and immune-protected, yet potentially druggable, cancer.

Biomarkers Identified for Methamphetamine-Induced Psychosis in Whole-Genome Expression Profiling Study

Methamphetamine stimulates the central nervous system and affects several pathways in different organs. Methamphetamine use disorder changes behavioral characteristics, which in some cases, includes psychotic symptoms. Mechanisms of psychosis in meth-induced psychosis and its similarities and differences with psychosis in individuals with schizophrenia are not clear. The present study aimed to shed light on molecular mechanisms which were affected in methamphetamine-induced psychosis by using whole-genome expressions profiling in the postmortem prefrontal cortex (PFC) and live subjects’ blood along with clinical evaluation. The study was included chronic persons with methamphetamine use disorder without psychoses, chronic persons with methamphetamine use disorder with Meth-induced psychosis, schizophrenic patients, and non-psychiatric subjects. Each group was included 15 postmortem PFC tissue and 750 live subjects. RNA extracted and cDNA has been synthesized. Genome expression profiling was conducted by using the Affymetrix GeneChip Human Genome U133 plus 2.0 Array Platform and confirmed by qPCR. Clinical and demographic data were obtained from the subjects. Clinical assessements performed by PANSS and BPRS test in live subjects. Results indicate several genes expression changes in persons with methamphetamine use disorders, Meth-induced psychosis, and schizophrenic patients. Alteration of number of gene ontologies and pathways associated to immune system and mitochondria were common only in methamphetamine induced psychosis and schizophrenia. Some altered genes related to serotonergic synapse and metabolic pathways, changed only in Meth-induced psychotic patients. Also, several correlations were found between positive, negative and general psychiatric symptoms with the gene expression alterations in Meth-induced psychosis. Results suggest that there are hundreds of genes wich play role in meth-induced psychosis that has similarities and differences with genes involved in psychosis in schizophrenia. Findings could suggest new potential biomarkers for meth-psychoses as well as an extension of our knowledge about the molecular mechanisms of a very complicated mental situation wich called psychosis. Trial Registration: Registration number: GR34661. Funding: The study was funded neuroimaging genetic laboratory of Arvin gene Company and Islamic Azad university. Declaration of Interest: The authors declare that they have nothing to disclose. Ethical Approval: Study levels and the consent form were approved by the central ethical committee of Islamic Azad University based on Helsinki declaration obligations.

Culture Medium and Sex Drive Epigenetic Reprogramming in Preimplantation Bovine Embryos.

Assisted reproductive technologies impact transcriptome and epigenome of embryos and can result in long-term phenotypic consequences. Whole-genome DNA methylation profiles from individual bovine blastocysts in vivo- and in vitro-derived (using three sources of protein: reproductive fluids, blood serum and bovine serum albumin) were generated. The impact of in vitro culture on DNA methylation was analyzed, and sex-specific methylation differences at blastocyst stage were uncovered. In vivo embryos showed the highest levels of methylation (29.5%), close to those produced in vitro with serum, whilst embryos produced in vitro with reproductive fluids or albumin showed less global methylation (25–25.4%). During repetitive element analysis, the serum group was the most affected. DNA methylation differences between in vivo and in vitro groups were more frequent in the first intron than in CpGi in promoters. Moreover, hierarchical cluster analysis showed that sex produced a stronger bias in the results than embryo origin. For each group, distance between male and female embryos varied, with in vivo blastocyst showing a lesser distance. Between the sexually dimorphic methylated tiles, which were biased to X-chromosome, critical factors for reproduction, developmental process, cell proliferation and DNA methylation machinery were included. These results support the idea that blastocysts show sexually-dimorphic DNA methylation patterns, and the known picture about the blastocyst methylome should be reconsidered.

From Sampling to Sequencing: A Liquid Biopsy Pre-Analytic Workflow to Maximize Multi-Layer Genomic Information from a Single Tube.

Simple SummaryLiquid biopsies seek to isolate tumor derived genetic material that circulates in blood plasma or cerebrospinal fluid. The less-invasive character of liquid biopsies combined with the option for serial analyses bears enormous potential for treatment monitoring or surveillance. We aimed to establish robust sampling protocols and pre-analytical workflows to allow for site independent multi-layer liquid biopsy testing. For an optimal usage of precious material, we explored sample stabilization in various conservation tubes and describe a protocol for the parallel isolation of cell-free DNA and RNA. Quantification and quality control steps were optimized for minimal sample use with both high sensitivity and reproducibility. We provide detailed step-by-step information on how to i) choose the best-suited protocol and ii) implement this in the liquid biopsy workflow. We believe that our study has potential to increase comparability of liquid biopsy approaches to bring these one step closer to routine clinical application.Liquid biopsies hold great promise for the management of cancer. Reliable liquid biopsy data depend on stable and reproducible pre-analytical protocols that comply with quality measures, irrespective of the sampling and processing site. We established a workflow for plasma preservation, followed by processing, cell-free nucleic acid isolation, quantification, and enrichment of potentially tumor-derived cell-free DNA and RNA. Employing the same input material for a direct comparison of different kits and protocols allowed us to formulate unbiased recommendations for sample collection, storage, and processing. The presented workflow integrates the stabilization in Norgen, PAX, or Streck tubes and subsequent parallel isolation of cell-free DNA and RNA with NucleoSnap and NucleoSpin. Qubit, Bioanalyzer, and TapeStation quantification and quality control steps were optimized for minimal sample use and high sensitivity and reproducibility. We show the efficiency of the proposed workflow by successful droplet digital PCR amplification of both cell-free DNA and RNA and by detection of tumor-specific alterations in low-coverage whole-genome sequencing and DNA methylation profiling of plasma-derived cell-free DNA. For the first time, we demonstrated successful parallel extraction of cell-free DNA and RNA from plasma samples. This workflow paves the road towards multi-layer genomic analysis from one single liquid biopsy sample.

Comprehensive DNA Methylation-transcriptome Profiles Association Analysis During the Treatment of Acute Myelocytic Leukemia.

Epigenetic modifications have recently attracted much attention in the study of the biological mechanisms of Acute Myelocytic Leukemia (AML) for therapy and prognosis. However, studies on DNA methylation changes during AML treatment are limited. The comprehensive DNA methylation-transcriptome profiles association analysis in this study aimed to establish whole-genome DNA methylation profiles and explore DNA methylation-related genes and their potential functions before and after treatment. And more appropriate biomarkers are expected to be identified for therapy strategies in AML. Illumina 450K and RNA-Seq data were obtained from the Cancer Genome Atlas. We performed comprehensive DNA methylation-transcriptome profiles association analysis, pathway analysis, correlation analysis, and survival analyses. The StarBase database was utilized to predict interactions between lncRNAs, miRNAs and target mRNAs. In total, 1592 distinct CpG sites and 2419 different expression transcripts were identified between pretreatment and post-treatment AML. The significantly enriched functions of methylated genes were stem cell differentiation, cell population maintenance, and cell development. The expression of UGT3A2, MOG, and VSTM1 was correlated with DNA methylation levels (r2 >0.5). Lastly, we identified 4 lncRNAs, 9 miRNAs and 142 mRNAs to construct a lncRNA-miRNA-mRNA ceRNA network. Our results revealed that DNA methylation was altered before and after treatment. Alterations in DNA methylation affected target gene expression and participated in the key biological processes of AML. Therefore, ceRNA networks may provide further insight into the study of favorable therapeutic markers in AML.