Rod photoreceptors are specialized neurons that mediate vision in dim light and are the predominant photoreceptor type in nocturnal mammals. The rods of nocturnal mammals are unique among vertebrate cell types in having an ‘inverted’ nuclear architecture, with a dense mass of heterochromatin in the center of the nucleus rather than dispersed clumps at the periphery. To test if this unique nuclear architecture is correlated with a unique epigenomic landscape, we performed ATAC-seq on mouse rods and their most closely related cell type, cone photoreceptors. We find that thousands of loci are selectively closed in rods relative to cones as well as >60 additional cell types. Furthermore, we find that the open chromatin profile of photoreceptors lacking the rod master regulator Nrl is nearly indistinguishable from that of native cones, indicating that Nrl is required for selective chromatin closure in rods. Finally, we identified distinct enrichments of transcription factor binding sites in rods and cones, revealing key differences in the cis-regulatory grammar of these cell types. Taken together, these data provide insight into the development and maintenance of photoreceptor identity, and highlight rods as an attractive system for studying the relationship between nuclear organization and local changes in gene regulation.

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Cis-regulatory elements (CREs, e.g., promoters and enhancers) regulate gene expression, and variants within CREs can modulate disease risk. Next-generation sequencing has enabled the rapid generation of genomic data that predict the locations of CREs, but a bottleneck lies in functionally interpreting these data. To address this issue, massively parallel reporter assays (MPRAs) have emerged, in which barcoded reporter libraries are introduced into cells, and the resulting barcoded transcripts are quantified by next-generation sequencing. Thus far, MPRAs have been largely restricted to assaying short CREs in a limited repertoire of cultured cell types. Here, we present two advances that extend the biological relevance and applicability of MPRAs. First, we adapt exome capture technology to instead capture candidate CREs, thereby tiling across the targeted regions and markedly increasing the length of CREs that can be readily assayed. Second, we package the library into adeno-associated virus (AAV), thereby allowing delivery to target organs in vivo. As a proof of concept, we introduce a capture library of about 46,000 constructs, corresponding to roughly 3500 DNase I hypersensitive (DHS) sites, into the mouse retina by ex vivo plasmid electroporation and into the mouse cerebral cortex by in vivo AAV injection. We demonstrate tissue-specific cis-regulatory activity of DHSs and provide examples of high-resolution truncation mutation analysis for multiplex parsing of CREs. Our approach should enable massively parallel functional analysis of a wide range of CREs in any organ or species that can be infected by AAV, such as nonhuman primates and human stem cell-derived organoids.

Next-generation sequencing has been used to infer the clonality of heterogeneous tumor samples. These analyses yield specific predictions—the population frequency of individual clones, their genetic composition, and their evolutionary relationships—which we set out to test by sequencing individual cells from three subjects diagnosed with secondary acute myeloid leukemia, each of whom had been previously characterized by whole genome sequencing of unfractionated tumor samples. Single-cell mutation profiling strongly supported the clonal architecture implied by the analysis of bulk material. In addition, it resolved the clonal assignment of single nucleotide variants that had been initially ambiguous and identified areas of previously unappreciated complexity. Accordingly, we find that many of the key assumptions underlying the analysis of tumor clonality by deep sequencing of unfractionated material are valid. Furthermore, we illustrate a single-cell sequencing strategy for interrogating the clonal relationships among known variants that is cost-effective, scalable, and adaptable to the analysis of both hematopoietic and solid tumors, or any heterogeneous population of cells.

Infant leukemia (IL) is a rare sporadic cancer with a grim prognosis. While most cases are accompanied by MLL-rearrangements and harbor very few somatic mutations; less is known about the genetics of the cases without MLL translocations. We performed the largest exome sequencing study to date on matched non-cancer DNA from pairs of mothers and IL patients to characterize congenital variation that may contribute to early leukemogenesis. Using the COSMIC database to define acute leukemia-associated candidate genes, we find a significant enrichment of rare, potentially functional congenital variation in IL patients compared to randomly selected genes within the same patients and unaffected pediatric controls. IL AML patients had more overall variation than IL ALL patients, but less of that variation was inherited from mothers. Of our candidate genes, we found that MLL3 was a compound heterozygote in every infant who developed AML and 50% of infants who developed ALL. These data suggest a model by which known genetic mechanisms for leukemogenesis could be disrupted without an abundance of somatic mutation or chromosomal rearrangements. This model would be consistent with existing models for the establishment of leukemia clones in utero and the high rate of IL concordance in monozygotic twins.

Thyroid hormone (TH) signaling plays an important role in the regulation of long-wavelength vision in vertebrates. In the retina,thyroid hormone receptor β(thrb) is required for expression of long-wavelength-sensitive opsin (lws) in red cone photoreceptors, while in retinal pigment epithelium (RPE), TH regulates expression of a cytochrome P450 enzyme,cyp27c1, that converts vitamin A1into vitamin A2to produce a red-shifted chromophore. To better understand how TH controls these processes, we analyzed the phenotype of zebrafish with mutations in the three known TH nuclear receptor transcription factors (thraa,thrab,and thrb). We found that no single TH nuclear receptor is required for TH-mediated induction ofcyp27c1but that deletion of all three (thraa−/−;thrab−/−;thrb−/−) completely abrogates its induction and the resulting conversion of A1- to A2-based retinoids. In the retina, loss ofthrbresulted in an absence of red cones at both larval and adult stages without disruption of the underlying cone mosaic. RNA-sequencing analysis revealed significant down-regulation of only five genes in adultthrb−/−retina, of which three (lws1,lws2, andmiR-726) occur in a single syntenic cluster. In thethrb−/−retina, retinal progenitors destined to become red cones were transfated into ultraviolet (UV) cones and horizontal cells. Taken together, our findings demonstrate cooperative regulation ofcyp27c1by TH receptors and a requirement forthrbin red cone fate determination. Thus, TH signaling coordinately regulates both spectral sensitivity and sensory plasticity.

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BackgroundRare genetic variation in the human population is a major source of pathophysiological variability and has been implicated in a host of complex phenotypes and diseases. Finding disease-related genes harboring disparate functional rare variants requires sequencing of many individuals across many genomic regions and comparing against unaffected cohorts. However, despite persistent declines in sequencing costs, population-based rare variant detection across large genomic target regions remains cost prohibitive for most investigators. In addition, DNA samples are often precious and hybridization methods typically require large amounts of input DNA. Pooled sample DNA sequencing is a cost and time-efficient strategy for surveying populations of individuals for rare variants. We set out to 1) create a scalable, multiplexing method for custom capture with or without individual DNA indexing that was amenable to low amounts of input DNA and 2) expand the functionality of the SPLINTER algorithm for calling substitutions, insertions and deletions across either candidate genes or the entire exome by integrating the variant calling algorithm with the dynamic programming aligner, Novoalign.ResultsWe report methodology for pooled hybridization capture with pre-enrichment, indexed multiplexing of up to 48 individuals or non-indexed pooled sequencing of up to 92 individuals with as little as 70 ng of DNA per person. Modified solid phase reversible immobilization bead purification strategies enable no sample transfers from sonication in 96-well plates through adapter ligation, resulting in 50% less library preparation reagent consumption. Custom Y-shaped adapters containing novel 7 base pair index sequences with a Hamming distance of ≥2 were directly ligated onto fragmented source DNA eliminating the need for PCR to incorporate indexes, and was followed by a custom blocking strategy using a single oligonucleotide regardless of index sequence. These results were obtained aligning raw reads against the entire genome using Novoalign followed by variant calling of non-indexed pools using SPLINTER or SAMtools for indexed samples. With these pipelines, we find sensitivity and specificity of 99.4% and 99.7% for pooled exome sequencing. Sensitivity, and to a lesser degree specificity, proved to be a function of coverage. For rare variants (≤2% minor allele frequency), we achieved sensitivity and specificity of ≥94.9% and ≥99.99% for custom capture of 2.5 Mb in multiplexed libraries of 22–48 individuals with only ≥5-fold coverage/chromosome, but these parameters improved to ≥98.7 and 100% with 20-fold coverage/chromosome.ConclusionsThis highly scalable methodology enables accurate rare variant detection, with or without individual DNA sample indexing, while reducing the amount of required source DNA and total costs through less hybridization reagent consumption, multi-sample sonication in a standard PCR plate, multiplexed pre-enrichment pooling with a single hybridization and lesser sequencing coverage required to obtain high sens...