Jeffrey A. Jeddeloh

Somatic retrotransposition alters the genetic landscape of the human brain

Retrotransposons are mobile genetic elements that use a germline ‘copy-and-paste’ mechanism to spread throughout metazoan genomes. At least 50 per cent of the human genome is derived from retrotransposons, with three active families (L1, Alu and SVA) associated with insertional mutagenesis and disease. Epigenetic and post-transcriptional suppression block retrotransposition in somatic cells, excluding early embryo development and some malignancies. Recent reports of L1 expression and copy number variation in the human brain suggest that L1 mobilization may also occur during later development. However, the corresponding integration sites have not been mapped. Here we apply a high-throughput method to identify numerous L1, Alu and SVA germline mutations, as well as 7,743 putative somatic L1 insertions, in the hippocampus and caudate nucleus of three individuals. Surprisingly, we also found 13,692 somatic Alu insertions and 1,350 SVA insertions. Our results demonstrate that retrotransposons mobilize to protein-coding genes differentially expressed and active in the brain. Thus, somatic genome mosaicism driven by retrotransposition may reshape the genetic circuitry that underpins normal and abnormal neurobiological processes.

Maize inbreds exhibit high levels of copy number variation (CNV) and presence/absence variation (PAV) in genome content

Following the domestication of maize over the past ∼10,000 years, breeders have exploited the extensive genetic diversity of this species to mold its phenotype to meet human needs. The extent of structural variation, including copy number variation (CNV) and presence/absence variation (PAV), which are thought to contribute to the extraordinary phenotypic diversity and plasticity of this important crop, have not been elucidated. Whole-genome, array-based, comparative genomic hybridization (CGH) revealed a level of structural diversity between the inbred lines B73 and Mo17 that is unprecedented among higher eukaryotes. A detailed analysis of altered segments of DNA conservatively estimates that there are several hundred CNV sequences among the two genotypes, as well as several thousand PAV sequences that are present in B73 but not Mo17. Haplotype-specific PAVs contain hundreds of single-copy, expressed genes that may contribute to heterosis and to the extraordinary phenotypic diversity of this important crop.

Targeted RNA sequencing reveals the deep complexity of the human transcriptome

Transcriptomic analyses have revealed an unexpected complexity to the human transcriptome, whose breadth and depth exceeds current RNA sequencing capability. Using tiling arrays to target and sequence select portions of the transcriptome, we identify and characterize unannotated transcripts whose rare or transient expression is below the detection limits of conventional sequencing approaches. We use the unprecedented depth of coverage afforded by this technique to reach the deepest limits of the human transcriptome, exposing widespread, regulated and remarkably complex noncoding transcription in intergenic regions, as well as unannotated exons and splicing patterns in even intensively studied protein-coding loci such as p53 and HOX. The data also show that intermittent sequenced reads observed in conventional RNA sequencing data sets, previously dismissed as noise, are in fact indicative of unassembled rare transcripts. Collectively, these results reveal the range, depth and complexity of a human transcriptome that is far from fully characterized.

Sorghum genome sequencing by methylation filtration.

Sorghum bicolor is a close relative of maize and is a staple crop in Africa and much of the developing world because of its superior tolerance of arid growth conditions. We have generated sequence from the hypomethylated portion of the sorghum genome by applying methylation filtration (MF) technology. The evidence suggests that 96% of the genes have been sequence tagged, with an average coverage of 65% across their length. Remarkably, this level of gene discovery was accomplished after generating a raw coverage of less than 300 megabases of the 735-megabase genome. MF preferentially captures exons and introns, promoters, microRNAs, and simple sequence repeats, and minimizes interspersed repeats, thus providing a robust view of the functional parts of the genome. The sorghum MF sequence set is beneficial to research on sorghum and is also a powerful resource for comparative genomics among the grasses and across the entire plant kingdom. Thousands of hypothetical gene predictions in rice and Arabidopsis are supported by the sorghum dataset, and genomic similarities highlight evolutionarily conserved regions that will lead to a better understanding of rice and Arabidopsis.

Whole exome capture in solution with 3 Gbp of data.

We have developed a solution-based method for targeted DNA capture-sequencing that is directed to the complete human exome. Using this approach allows the discovery of greater than 95% of all expected heterozygous singe base variants, requires as little as 3 Gbp of raw sequence data and constitutes an effective tool for identifying rare coding alleles in large scale genomic studies.

Barley whole exome capture: a tool for genomic research in the genus Hordeum and beyond

Advanced resources for genome-assisted research in barley (Hordeum vulgare) including a whole-genome shotgun assembly and an integrated physical map have recently become available. These have made possible studies that aim to assess genetic diversity or to isolate single genes by whole-genome resequencing and in silico variant detection. However such an approach remains expensive given the 5 Gb size of the barley genome. Targeted sequencing of the mRNA-coding exome reduces barley genomic complexity more than 50-fold, thus dramatically reducing this heavy sequencing and analysis load. We have developed and employed an in-solution hybridization-based sequence capture platform to selectively enrich for a 61.6 megabase coding sequence target that includes predicted genes from the genome assembly of the cultivar Morex as well as publicly available full-length cDNAs and de novo assembled RNA-Seq consensus sequence contigs. The platform provides a highly specific capture with substantial and reproducible enrichment of targeted exons, both for cultivated barley and related species. We show that this exome capture platform provides a clear path towards a broader and deeper understanding of the natural variation residing in the mRNA-coding part of the barley genome and will thus constitute a valuable resource for applications such as mapping-by-sequencing and genetic diversity analyzes.

Structural variants in the soybean genome localize to clusters of biotic stress response genes

Genome-wide structural and gene content variations are hypothesized to drive important phenotypic variation within a species. Structural and gene content variations were assessed among four soybean (Glycine max) genotypes using array hybridization and targeted resequencing. Many chromosomes exhibited relatively low rates of structural variation (SV) among genotypes. However, several regions exhibited both copy number and presence-absence variation, the most prominent found on chromosomes 3, 6, 7, 16, and 18. Interestingly, the regions most enriched for SV were specifically localized to gene-rich regions that harbor clustered multigene families. The most abundant classes of gene families associated with these regions were the nucleotide-binding and receptor-like protein classes, both of which are important for plant biotic defense. The colocalization of SV with plant defense response signal transduction pathways provides insight into the mechanisms of soybean resistance gene evolution and may inform the development of new approaches to resistance gene cloning.

Phenotypic and genomic analyses of a fast neutron mutant population resource in soybean

Mutagenized populations have become indispensable resources for introducing variation and studying gene function in plant genomics research. In this study, fast neutron (FN) radiation was used to induce deletion mutations in the soybean (Glycine max) genome. Approximately 120,000 soybean seeds were exposed to FN radiation doses of up to 32 Gray units to develop over 23,000 independent M2 lines. Here, we demonstrate the utility of this population for phenotypic screening and associated genomic characterization of striking and agronomically important traits. Plant variation was cataloged for seed composition, maturity, morphology, pigmentation, and nodulation traits. Mutants that showed significant increases or decreases in seed protein and oil content across multiple generations and environments were identified. The application of comparative genomic hybridization (CGH) to lesion-induced mutants for deletion mapping was validated on a midoleate x-ray mutant, M23, with a known FAD2-1A (for fatty acid desaturase) gene deletion. Using CGH, a subset of mutants was characterized, revealing deletion regions and candidate genes associated with phenotypes of interest. Exome resequencing and sequencing of PCR products confirmed FN-induced deletions detected by CGH. Beyond characterization of soybean FN mutants, this study demonstrates the utility of CGH, exome sequence capture, and next-generation sequencing approaches for analyses of mutant plant genomes. We present this FN mutant soybean population as a valuable public resource for future genetic screens and functional genomics research.

The composition and origins of genomic variation among individuals of the soybean reference cultivar Williams 82

Soybean (Glycine max) is a self-pollinating species that has relatively low nucleotide polymorphism rates compared with other crop species. Despite the low rate of nucleotide polymorphisms, a wide range of heritable phenotypic variation exists. There is even evidence for heritable phenotypic variation among individuals within some cultivars. Williams 82, the soybean cultivar used to produce the reference genome sequence, was derived from backcrossing a Phytophthora root rot resistance locus from the donor parent Kingwa into the recurrent parent Williams. To explore the genetic basis of intracultivar variation, we investigated the nucleotide, structural, and gene content variation of different Williams 82 individuals. Williams 82 individuals exhibited variation in the number and size of introgressed Kingwa loci. In these regions of genomic heterogeneity, the reference Williams 82 genome sequence consists of a mosaic of Williams and Kingwa haplotypes. Genomic structural variation between Williams and Kingwa was maintained between the Williams 82 individuals within the regions of heterogeneity. Additionally, the regions of heterogeneity exhibited gene content differences between Williams 82 individuals. These findings show that genetic heterogeneity in Williams 82 primarily originated from the differential segregation of polymorphic chromosomal regions following the backcross and single-seed descent generations of the breeding process. We conclude that soybean haplotypes can possess a high rate of structural and gene content variation, and the impact of intracultivar genetic heterogeneity may be significant. This detailed characterization will be useful for interpreting soybean genomic data sets and highlights important considerations for research communities that are developing or utilizing a reference genome sequence.

Identification of Novel High-Frequency DNA Methylation Changes in Breast Cancer

Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis. The inherent thermodynamic stability of cytosine methylation and the apparent high specificity of the alterations for disease may accelerate the development of powerful molecular diagnostics for cancer. We report a genome-wide analysis of DNA methylation alterations in breast cancer. The approach efficiently identified a large collection of novel differentially DNA methylated loci (∼200), a subset of which was independently validated across a panel of over 230 clinical samples. The differential cytosine methylation events were independent of patient age, tumor stage, estrogen receptor status or family history of breast cancer. The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively. Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date. The discovery of over 50 novel DNA methylation-based biomarkers of breast cancer may provide new routes for development of DNA methylation-based diagnostics and prognostics, as well as reveal epigenetically regulated mechanism involved in breast tumorigenesis.