Mechanisms of establishment and functional significance of DNA demethylation during erythroid differentiation.

Erythroid differentiation is associated with global DNA demethylation, but a complete methylome was lacking in the erythroid lineage. We have generated allele-specific base resolution methylomes of primary basophilic erythroblasts (BasoEs) and compared these with 8 other cell types. We found that DNA demethylation during differentiation from hematopoietic stem/progenitor cells (HSPCs) to BasoEs occurred predominantly in intergenic sequences and in inactive gene bodies causing the formation of partially methylated domains (PMDs) in 74% of the BasoE methylome. Moreover, differentially methylated regions (DMRs) between HSPCs and BasoEs occurred mostly in putative enhancer regions and were most often associated with GATA, EKLF, and AP1 binding motifs. Surprisingly, promoters silent in both HSPCs and BasoEs exhibited much more dramatic chromatin changes during differentiation than activated promoters. Unmethylated silent promoters were often associated with active chromatin states in highly methylated domains (HMDs) but with polycomb-repression in PMDs, indicating that silent promoters are generally regulated differently in HMDs and PMDs. We show that long PMDs replicate late, but that short PMDs replicate early and therefore that the partial methylation of DNA after replication during erythroid expansion occurs throughout S phase of the cell cycle. We propose that baseline maintenance methylation following replication decreases during erythroid differentiation resulting in PMD formation and that the presence of HMDs in the BasoE methylome results from transcription-associated DNA methylation of gene bodies. We detected ∼700 large allele-specific DMRs that were enriched in single-nucleotide polymorphisms, suggesting that primary DNA sequence might be a determinant of DNA methylation levels within PMDs.

Allele-specific analysis of DNA replication origins in mammalian cells.

The mechanisms that control the location and timing of firing of replication origins are poorly understood. Using a novel functional genomic approach based on the analysis of SNPs and indels in phased human genomes, we observe that replication asynchrony is associated with small cumulative variations in the initiation efficiency of multiple origins between the chromosome homologues, rather than with the activation of dormant origins. Allele-specific measurements demonstrate that the presence of G-quadruplex-forming sequences does not correlate with the efficiency of initiation. Sequence analysis reveals that the origins are highly enriched in sequences with profoundly asymmetric G/C and A/T nucleotide distributions and are almost completely depleted of antiparallel triplex-forming sequences. We therefore propose that although G4-forming sequences are abundant in replication origins, an asymmetry in nucleotide distribution, which increases the propensity of origins to unwind and adopt non-B DNA structure, rather than the ability to form G4, is directly associated with origin activity.

GenPlay Multi-Genome, a tool to compare and analyze multiple human genomes in a graphical interface.

SUMMARY: Parallel visualization of multiple individual human genomes is a complex endeavor that is rapidly gaining importance with the increasing number of personal, phased and cancer genomes that are being generated. It requires the display of variants such as SNPs, indels and structural variants that are unique to specific genomes and the introduction of multiple overlapping gaps in the reference sequence. Here, we describe GenPlay Multi-Genome, an application specifically written to visualize and analyze multiple human genomes in parallel. GenPlay Multi-Genome is ideally suited for the comparison of allele-specific expression and functional genomic data obtained from multiple phased genomes in a graphical interface with access to multiple-track operation. It also allows the analysis of data that have been aligned to custom genomes rather than to a standard reference and can be used as a variant calling format file browser and as a tool to compare different genome assembly, such as hg19 and hg38. AVAILABILITY AND IMPLEMENTATION: GenPlay is available under the GNU public license (GPL-3) from http://genplay.einstein.yu.edu. The source code is available at https://github.com/JulienLajugie/GenPlay.

Allele-specific genome-wide profiling in human primary erythroblasts reveal replication program organization.

We have developed a new approach to characterize allele-specific timing of DNA replication genome-wide in human primary basophilic erythroblasts. We show that the two chromosome homologs replicate at the same time in about 88% of the genome and that large structural variants are preferentially associated with asynchronous replication. We identified about 600 megabase-sized asynchronously replicated domains in two tested individuals. The longest asynchronously replicated domains are enriched in imprinted genes suggesting that structural variants and parental imprinting are two causes of replication asynchrony in the human genome. Biased chromosome X inactivation in one of the two individuals tested was another source of detectable replication asynchrony. Analysis of high-resolution TimEX profiles revealed small variations termed timing ripples, which were undetected in previous, lower resolution analyses. Timing ripples reflect highly reproducible, variations of the timing of replication in the 100 kb-range that exist within the well-characterized megabase-sized replication timing domains. These ripples correspond to clusters of origins of replication that we detected using novel nascent strands DNA profiling methods. Analysis of the distribution of replication origins revealed dramatic differences in initiation of replication frequencies during S phase and a strong association, in both synchronous and asynchronous regions, between origins of replication and three genomic features: G-quadruplexes, CpG Islands and transcription start sites. The frequency of initiation in asynchronous regions was similar in the two homologs. Asynchronous regions were richer in origins of replication than synchronous regions.

Complete genome phasing of family quartet by combination of genetic, physical and population-based phasing analysis.

Phased genome maps are important to understand genetic and epigenetic regulation and disease mechanisms, particularly parental imprinting defects. Phasing is also critical to assess the functional consequences of genetic variants, and to allow precise definition of haplotype blocks which is useful to understand gene-flow and genotype-phenotype association at the population level. Transmission phasing by analysis of a family quartet allows the phasing of 95% of all variants as the uniformly heterozygous positions cannot be phased. Here, we report a phasing method based on a combination of transmission analysis, physical phasing by pair-end sequencing of libraries of staggered sizes and population-based analysis. Sequencing of a healthy Caucasians quartet at 120x coverage and combination of physical and transmission phasing yielded the phased genotypes of about 99.8% of the SNPs, indels and structural variants present in the quartet, a phasing rate significantly higher than what can be achieved using any single phasing method. A false positive SNP error rate below 10*E-7 per genome and per base was obtained using a combination of filters. We provide a complete list of SNPs, indels and structural variants, an analysis of haplotype block sizes, and an analysis of the false positive and negative variant calling error rates. Improved genome phasing and family sequencing will increase the power of genome-wide sequencing as a clinical diagnosis tool and has myriad basic science applications.

GenPlay, a multipurpose genome analyzer and browser.

MOTIVATION: Rapidly decreasing sequencing cost due to the emergence and improvement of massively parallel sequencing technologies has resulted in a dramatic increase in the quantity of data that needs to be analyzed. Therefore, software tools to process, visualize, analyze and integrate data produced on multiple platforms and using multiple methods are needed. RESULTS: GenPlay is a fast, easy to use and stable tool for rapid analysis and data processing. It is written in Java and runs on all major operating systems. GenPlay recognizes a wide variety of common genomic data formats from microarray- or sequencing-based platforms and offers a library of operations (normalization, binning, smoothing) to process raw data into visualizable tracks. GenPlay displays tracks adapted to summarize gene structure, gene expression, repeat families, CPG islands, etc. as well as custom tracks to show the results of RNA-Seq, ChIP-Seq, TimEX-Seq and single nucleotide polymorphism (SNP) analysis. GenPlay can generate statistics (minimum, maximum, SD, correlation, etc.). The tools provided include Gaussian filter, peak finders, signal saturation, island finders. The software also offers graphical features such as scatter plots and bar charts to depict signal repartition. The library of operations is continuously growing based on the emerging needs. AVAILABILITY: GenPlay is an open-source project available from http://www.genplay.net. The code source of the software is available at https://genplay.einstein.yu.edu/svn/GenPlay.

Predictable dynamic program of timing of DNA replication in human cells.

The organization of mammalian DNA replication is poorly understood. We have produced high-resolution dynamic maps of the timing of replication in human erythroid, mesenchymal, and embryonic stem (ES) cells using TimEX, a method that relies on gaussian convolution of massive, highly redundant determinations of DNA copy-number variations during S phase to produce replication timing profiles. We first obtained timing maps of 3% of the genome using high-density oligonucleotide tiling arrays and then extended the TimEX method genome-wide using massively parallel sequencing. We show that in untransformed human cells, timing of replication is highly regulated and highly synchronous, and that many genomic segments are replicated in temporal transition regions devoid of initiation, where replication forks progress unidirectionally from origins that can be hundreds of kilobases away. Absence of initiation in one transition region is shown at the molecular level by single molecule analysis of replicated DNA (SMARD). Comparison of ES and erythroid cells replication patterns revealed that these cells replicate about 20% of their genome in different quarters of S phase. Importantly, we detected a strong inverse relationship between timing of replication and distance to the closest expressed gene. This relationship can be used to predict tissue-specific timing of replication profiles from expression data and genomic annotations. We also provide evidence that early origins of replication are preferentially located near highly expressed genes, that mid-firing origins are located near moderately expressed genes, and that late-firing origins are located far from genes.