High-resolution mapping and characterization of open chromatin across the genome.

Mapping DNase I hypersensitive (HS) sites is an accurate method of identifying the location of genetic regulatory elements, including promoters, enhancers, silencers, insulators, and locus control regions. We employed high-throughput sequencing and whole-genome tiled array strategies to identify DNase I HS sites within human primary CD4+ T cells. Combining these two technologies, we have created a comprehensive and accurate genome-wide open chromatin map. Surprisingly, only 16%-21% of the identified 94,925 DNase I HS sites are found in promoters or first exons of known genes, but nearly half of the most open sites are in these regions. In conjunction with expression, motif, and chromatin immunoprecipitation data, we find evidence of cell-type-specific characteristics, including the ability to identify transcription start sites and locations of different chromatin marks utilized in these cells. In addition, and unexpectedly, our analyses have uncovered detailed features of nucleosome structure.

A reference data set of 5.4 million phased human variants validated by genetic inheritance from sequencing a three-generation 17-member pedigree.

Improvement of variant calling in next-generation sequence data requires a comprehensive, genome-wide catalog of high-confidence variants called in a set of genomes for use as a benchmark. We generated deep, whole-genome sequence data of 17 individuals in a three-generation pedigree and called variants in each genome using a range of currently available algorithms. We used haplotype transmission information to create a phased "Platinum" variant catalog of 4.7 million single-nucleotide variants (SNVs) plus 0.7 million small (1-50 bp) insertions and deletions (indels) that are consistent with the pattern of inheritance in the parents and 11 children of this pedigree. Platinum genotypes are highly concordant with the current catalog of the National Institute of Standards and Technology for both SNVs (>99.99%) and indels (99.92%) and add a validated truth catalog that has 26% more SNVs and 45% more indels. Analysis of 334,652 SNVs that were consistent between informatics pipelines yet inconsistent with haplotype transmission ("nonplatinum") revealed that the majority of these variants are de novo and cell-line mutations or reside within previously unidentified duplications and deletions. The reference materials from this study are a resource for objective assessment of the accuracy of variant calls throughout genomes.

Sequencing studies in human genetics: design and interpretation.

Next-generation sequencing is becoming the primary discovery tool in human genetics. There have been many clear successes in identifying genes that are responsible for Mendelian diseases, and sequencing approaches are now poised to identify the mutations that cause undiagnosed childhood genetic diseases and those that predispose individuals to more common complex diseases. There are, however, growing concerns that the complexity and magnitude of complete sequence data could lead to an explosion of weakly justified claims of association between genetic variants and disease. Here, we provide an overview of the basic workflow in next-generation sequencing studies and emphasize, where possible, measures and considerations that facilitate accurate inferences from human sequencing studies.

Adult pallium transcriptomes surprise in not reflecting predicted homologies across diverse chicken and mouse pallial sectors.

The thorniest problem in comparative neurobiology is the identification of the particular brain region of birds and reptiles that corresponds to the mammalian neocortex [Butler AB, Reiner A, Karten HJ (2011) Ann N Y Acad Sci 1225:14-27; Wang Y, Brzozowska-Prechtl A, Karten HJ (2010) Proc Natl Acad Sci USA 107(28):12676-12681]. We explored which genes are actively transcribed in the regions of controversial ancestry in a representative bird (chicken) and mammal (mouse) at adult stages. We conducted four analyses comparing the expression patterns of their 5,130 most highly expressed one-to-one orthologous genes that considered global patterns of expression specificity, strong gene markers, and coexpression networks. Our study demonstrates transcriptomic divergence, plausible convergence, and, in two exceptional cases, conservation between specialized avian and mammalian telencephalic regions. This large-scale study potentially resolves the complex relationship between developmental homology and functional characteristics on the molecular level and settles long-standing evolutionary debates.

Whole-genome sequencing identifies a recurrent functional synonymous mutation in melanoma.

Synonymous mutations, which do not alter the protein sequence, have been shown to affect protein function [Sauna ZE, Kimchi-Sarfaty C (2011) Nat Rev Genet 12(10):683-691]. However, synonymous mutations are rarely investigated in the cancer genomics field. We used whole-genome and -exome sequencing to identify somatic mutations in 29 melanoma samples. Validation of one synonymous somatic mutation in BCL2L12 in 285 samples identified 12 cases that harbored the recurrent F17F mutation. This mutation led to increased BCL2L12 mRNA and protein levels because of differential targeting of WT and mutant BCL2L12 by hsa-miR-671-5p. Protein made from mutant BCL2L12 transcript bound p53, inhibited UV-induced apoptosis more efficiently than WT BCL2L12, and reduced endogenous p53 target gene transcription. This report shows selection of a recurrent somatic synonymous mutation in cancer. Our data indicate that silent alterations have a role to play in human cancer, emphasizing the importance of their investigation in future cancer genome studies.

Genome-wide DNA methylation profiles in hematopoietic stem and progenitor cells reveal overrepresentation of ETS transcription factor binding sites.

DNA methylation is an essential epigenetic mark that is required for normal development. Knockout of the DNA methyltransferase enzymes in the mouse hematopoietic compartment reveals that methylation is critical for hematopoietic differentiation. To better understand the role of DNA methylation in hematopoiesis, we characterized genome-wide DNA methylation in primary mouse hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), and erythroblasts (ERYs). Methyl binding domain protein 2 (MBD) enrichment of DNA followed by massively parallel sequencing (MBD-seq) was used to map genome-wide DNA methylation. Globally, DNA methylation was most abundant in HSCs, with a 40% reduction in CMPs, and a 67% reduction in ERYs. Only 3% of peaks arise during differentiation, demonstrating a genome-wide decline in DNA methylation during erythroid development. Analysis of genomic features revealed that 98% of promoter CpG islands are hypomethylated, while 20%-25% of non-promoter CpG islands are methylated. Proximal promoter sequences of expressed genes are hypomethylated in all cell types, while gene body methylation positively correlates with gene expression in HSCs and CMPs. Elevated genome-wide DNA methylation in HSCs and the positive association between methylation and gene expression demonstrates that DNA methylation is a mark of cellular plasticity in HSCs. Using de novo motif discovery, we identified overrepresented transcription factor consensus binding motifs in methylated sequences. Motifs for several ETS transcription factors, including GABPA and ELF1, are overrepresented in methylated regions. Our genome-wide survey demonstrates that DNA methylation is markedly altered during myeloid differentiation and identifies critical regions of the genome and transcription factor programs that contribute to hematopoiesis.

Mutational signatures of de-differentiation in functional non-coding regions of melanoma genomes.

Much emphasis has been placed on the identification, functional characterization, and therapeutic potential of somatic variants in tumor genomes. However, the majority of somatic variants lie outside coding regions and their role in cancer progression remains to be determined. In order to establish a system to test the functional importance of non-coding somatic variants in cancer, we created a low-passage cell culture of a metastatic melanoma tumor sample. As a foundation for interpreting functional assays, we performed whole-genome sequencing and analysis of this cell culture, the metastatic tumor from which it was derived, and the patient-matched normal genomes. When comparing somatic mutations identified in the cell culture and tissue genomes, we observe concordance at the majority of single nucleotide variants, whereas copy number changes are more variable. To understand the functional impact of non-coding somatic variation, we leveraged functional data generated by the ENCODE Project Consortium. We analyzed regulatory regions derived from multiple different cell types and found that melanocyte-specific regions are among the most depleted for somatic mutation accumulation. Significant depletion in other cell types suggests the metastatic melanoma cells de-differentiated to a more basal regulatory state. Experimental identification of genome-wide regulatory sites in two different melanoma samples supports this observation. Together, these results show that mutation accumulation in metastatic melanoma is nonrandom across the genome and that a de-differentiated regulatory architecture is common among different samples. Our findings enable identification of the underlying genetic components of melanoma and define the differences between a tissue-derived tumor sample and the cell culture created from it. Such information helps establish a broader mechanistic understanding of the linkage between non-coding genomic variations and the cellular evolution of cancer.

Extensive evolutionary changes in regulatory element activity during human origins are associated with altered gene expression and positive selection.

Understanding the molecular basis for phenotypic differences between humans and other primates remains an outstanding challenge. Mutations in non-coding regulatory DNA that alter gene expression have been hypothesized as a key driver of these phenotypic differences. This has been supported by differential gene expression analyses in general, but not by the identification of specific regulatory elements responsible for changes in transcription and phenotype. To identify the genetic source of regulatory differences, we mapped DNaseI hypersensitive (DHS) sites, which mark all types of active gene regulatory elements, genome-wide in the same cell type isolated from human, chimpanzee, and macaque. Most DHS sites were conserved among all three species, as expected based on their central role in regulating transcription. However, we found evidence that several hundred DHS sites were gained or lost on the lineages leading to modern human and chimpanzee. Species-specific DHS site gains are enriched near differentially expressed genes, are positively correlated with increased transcription, show evidence of branch-specific positive selection, and overlap with active chromatin marks. Species-specific sequence differences in transcription factor motifs found within these DHS sites are linked with species-specific changes in chromatin accessibility. Together, these indicate that the regulatory elements identified here are genetic contributors to transcriptional and phenotypic differences among primate species.

Accurate and comprehensive sequencing of personal genomes.

As whole-genome sequencing becomes commoditized and we begin to sequence and analyze personal genomes for clinical and diagnostic purposes, it is necessary to understand what constitutes a complete sequencing experiment for determining genotypes and detecting single-nucleotide variants. Here, we show that the current recommendation of ∼30× coverage is not adequate to produce genotype calls across a large fraction of the genome with acceptably low error rates. Our results are based on analyses of a clinical sample sequenced on two related Illumina platforms, GAII(x) and HiSeq 2000, to a very high depth (126×). We used these data to establish genotype-calling filters that dramatically increase accuracy. We also empirically determined how the callable portion of the genome varies as a function of the amount of sequence data used. These results help provide a "sequencing guide" for future whole-genome sequencing decisions and metrics by which coverage statistics should be reported.

Isaac: ultra-fast whole-genome secondary analysis on Illumina sequencing platforms.

SUMMARY: An ultrafast DNA sequence aligner (Isaac Genome Alignment Software) that takes advantage of high-memory hardware (>48 GB) and variant caller (Isaac Variant Caller) have been developed. We demonstrate that our combined pipeline (Isaac) is four to five times faster than BWA + GATK on equivalent hardware, with comparable accuracy as measured by trio conflict rates and sensitivity. We further show that Isaac is effective in the detection of disease-causing variants and can easily/economically be run on commodity hardware. AVAILABILITY: Isaac has an open source license and can be obtained at https://github.com/sequencing.

Approaches to comparative sequence analysis: towards a functional view of vertebrate genomes.

The comparison of genomic sequences is now a common approach to identifying and characterizing functional regions in vertebrate genomes. However, for theoretical reasons and because of practical issues, the generation of these data sets is non-trivial and can have many pitfalls. We are currently seeing an explosion of comparative sequence data, the benefits and limitations of which need to be disseminated to the scientific community. This Review provides a critical overview of the different types of sequence data that are available for analysis and of contemporary comparative sequence analysis methods, highlighting both their strengths and limitations. Approaches to determining the biological significance of constrained sequence are also explored.

A new strategy for genome assembly using short sequence reads and reduced representation libraries.

We have developed a novel approach for using massively parallel short-read sequencing to generate fast and inexpensive de novo genomic assemblies comparable to those generated by capillary-based methods. The ultrashort (<100 base) sequences generated by this technology pose specific biological and computational challenges for de novo assembly of large genomes. To account for this, we devised a method for experimentally partitioning the genome using reduced representation (RR) libraries prior to assembly. We use two restriction enzymes independently to create a series of overlapping fragment libraries, each containing a tractable subset of the genome. Together, these libraries allow us to reassemble the entire genome without the need of a reference sequence. As proof of concept, we applied this approach to sequence and assembled the majority of the 125-Mb Drosophila melanogaster genome. We subsequently demonstrate the accuracy of our assembly method with meaningful comparisons against the current available D. melanogaster reference genome (dm3). The ease of assembly and accuracy for comparative genomics suggest that our approach will scale to future mammalian genome-sequencing efforts, saving both time and money without sacrificing quality.

Systematic comparison of three genomic enrichment methods for massively parallel DNA sequencing.

Massively parallel DNA sequencing technologies have greatly increased our ability to generate large amounts of sequencing data at a rapid pace. Several methods have been developed to enrich for genomic regions of interest for targeted sequencing. We have compared three of these methods: Molecular Inversion Probes (MIP), Solution Hybrid Selection (SHS), and Microarray-based Genomic Selection (MGS). Using HapMap DNA samples, we compared each of these methods with respect to their ability to capture an identical set of exons and evolutionarily conserved regions associated with 528 genes (2.61 Mb). For sequence analysis, we developed and used a novel Bayesian genotype-assigning algorithm, Most Probable Genotype (MPG). All three capture methods were effective, but sensitivities (percentage of targeted bases associated with high-quality genotypes) varied for an equivalent amount of pass-filtered sequence: for example, 70% (MIP), 84% (SHS), and 91% (MGS) for 400 Mb. In contrast, all methods yielded similar accuracies of >99.84% when compared to Infinium 1M SNP BeadChip-derived genotypes and >99.998% when compared to 30-fold coverage whole-genome shotgun sequencing data. We also observed a low false-positive rate with all three methods; of the heterozygous positions identified by each of the capture methods, >99.57% agreed with 1M SNP BeadChip, and >98.840% agreed with the whole-genome shotgun data. In addition, we successfully piloted the genomic enrichment of a set of 12 pooled samples via the MGS method using molecular bar codes. We find that these three genomic enrichment methods are highly accurate and practical, with sensitivities comparable to that of 30-fold coverage whole-genome shotgun data.

Genome-wide ChIP-Seq reveals a dramatic shift in the binding of the transcription factor erythroid Kruppel-like factor during erythrocyte differentiation.

Erythropoiesis is dependent on the activity of transcription factors, including the erythroid-specific erythroid Kruppel-like factor (EKLF). ChIP followed by massively parallel sequencing (ChIP-Seq) is a powerful, unbiased method to map trans-factor occupancy. We used ChIP-Seq to study the interactome of EKLF in mouse erythroid progenitor cells and more differentiated erythroblasts. We correlated these results with the nuclear distribution of EKLF, RNA-Seq analysis of the transcriptome, and the occupancy of other erythroid transcription factors. In progenitor cells, EKLF is found predominantly at the periphery of the nucleus, where EKLF primarily occupies the promoter regions of genes and acts as a transcriptional activator. In erythroblasts, EKLF is distributed throughout the nucleus, and erythroblast-specific EKLF occupancy is predominantly in intragenic regions. In progenitor cells, EKLF modulates general cell growth and cell cycle regulatory pathways, whereas in erythroblasts EKLF is associated with repression of these pathways. The EKLF interactome shows very little overlap with the interactomes of GATA1, GATA2, or TAL1, leading to a model in which EKLF directs programs that are independent of those regulated by the GATA factors or TAL1.

Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project.

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.

Human NPY promoter variation rs16147:T>C as a moderator of prefrontal NPY gene expression and negative affect.

Studies in humans and animals suggest a role for NPY in the mediation of behavioral stress responses. Here, we examined whether the NPY promoter variant rs16147:T>C is functional for expression of NPY in a brain region relevant for behavioral control, anxiety and depression, the anterior cingulate cortex. In silico analysis of DNA structural profile changes produced by rs16147 variation suggests allelic differences in protein binding at the rs16147 site. This was confirmed by electrophoretic mobility shift assay, demonstrating that the rs16147 C-allele has strongly reduced affinity for a yet unknown factor compared to the T-allele. Analyzing 107 human post-mortem brain samples we show that allelic variation at rs16147 contributes to regulation of NPY mRNA and peptide levels in this region. Specifically, the C-allele leads to increased gene expression. In agreement with the molecular findings, rs16147:T>C is associated with anxiety and depressive symptoms in 314 young adults via a gene x environment interaction with early childhood adversity, replicating the recent finding of rs16147-C as a risk factor for stress related psychopathology. Our results show the importance of rs16147:T>C for regulation of NPY gene expression and brain function.

Early history of mammals is elucidated with the ENCODE multiple species sequencing data.

Understanding the early evolution of placental mammals is one of the most challenging issues in mammalian phylogeny. Here, we addressed this question by using the sequence data of the ENCODE consortium, which include 1% of mammalian genomes in 18 species belonging to all main mammalian lineages. Phylogenetic reconstructions based on an unprecedented amount of coding sequences taken from 218 genes resulted in a highly supported tree placing the root of Placentalia between Afrotheria and Exafroplacentalia (Afrotheria hypothesis). This topology was validated by the phylogenetic analysis of a new class of genomic phylogenetic markers, the conserved noncoding sequences. Applying the tests of alternative topologies on the coding sequence dataset resulted in the rejection of the Atlantogenata hypothesis (Xenarthra grouping with Afrotheria), while this test rejected the second alternative scenario, the Epitheria hypothesis (Xenarthra at the base), when using the noncoding sequence dataset. Thus, the two datasets support the Afrotheria hypothesis; however, none can reject both of the remaining topological alternatives.

Life-history traits drive the evolutionary rates of mammalian coding and noncoding genomic elements.

A comprehensive phylogenetic framework is indispensable for investigating the evolution of genomic features in mammals as a whole, and particularly in humans. Using the ENCODE sequence data, we estimated mammalian neutral evolutionary rates and selective pressures acting on conserved coding and noncoding elements. We show that neutral evolutionary rates can be explained by the generation time (GT) hypothesis. Accordingly, primates (especially humans), having longer GTs than other mammals, display slower rates of neutral evolution. The evolution of constrained elements, particularly of nonsynonymous sites, is in agreement with the expectations of the nearly neutral theory of molecular evolution. We show that rates of nonsynonymous substitutions (dN) depend on the population size of a species. The results are robust to the exclusion of hypermutable CpG prone sites. The average rate of evolution in conserved noncoding sequences (CNCs) is 1.7 times higher than in nonsynonymous sites. Despite this, CNCs evolve at similar or even lower rates than nonsynonymous sites in the majority of basal branches of the eutherian tree. This observation could be the result of an overall gradual or, alternatively, lineage-specific relaxation of CNCs. The latter hypothesis was supported by the finding that 3 of the 20 longest CNCs displayed significant relaxation of individual branches. This observation may explain why the evolution of CNCs fits the expectations of the nearly neutral theory less well than the evolution of nonsynonymous sites.

Differences between pair-wise and multi-sequence alignment methods affect vertebrate genome comparisons.

Producing complete and accurate alignments of multiple genomic sequences is complex and prone to errors, especially with sequences generated from highly diverged species. In this article, we show that multi-sequence (as opposed to pair-wise) alignment methods are substantially better at aligning (or 'capturing') all of the available orthologous sequence from phylogenetically diverse vertebrates (i.e. those separated by relatively long branch lengths). Maximum gains are obtained only when sequences from many species are aligned. Such multi-sequence alignments contain significant amounts of exonic and highly conserved non-exonic sequences that are not captured in pair-wise alignments, thus illustrating the importance of the alignment method used for performing comparative genome analyses.

The relationship between fine scale DNA structure, GC content, and functional elements in 1% of the human genome.

GC content has been shown to be an important aspect of human genomic function. Extending beyond the scope of GC content alone, there is a class of regions in the genome that have especially high GC content and are enriched for the CG dinucleotide--called CpG islands. CpG islands have been linked to biologically functional genomic elements. DNA structure also contributes to biological function. Recent studies found that some DNA structural properties are correlated with CpG island functionality. Here, we use hydroxyl radical cleavage patterns as a measure of DNA structure, to explore the relationship between GC content and fine-scale DNA structure. We show that there is a positive correlation between GC content and the solvent-accessible structural properties of a DNA sequence, and that the strength of this correlation decreases as genomic resolution increases. We demonstrate that regions of the genome that have highly solvent-accessible DNA structure tend to overlap functional genomic elements. Our results suggest that fine-scale DNA structural properties that are encoded in the genome are important for biological function, and that the highly solvent-accessible nature of high GC content regions and some CpG islands may account for some of their functional properties.