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A new generation of scalable single cell whole genome sequencing (scWGS) methods allows unprecedented high resolution measurement of the evolutionary dynamics of cancer cell populations. Phylogenetic reconstruction is central to identifying sub-populations and distinguishing the mutational processes that gave rise to them. Existing phylogenetic tree building models do not scale to the tens of thousands of high resolution genomes achievable with current scWGS methods. We constructed a phylogenetic model and associated Bayesian inference procedure, sitka, specifically for scWGS data. The method is based on a novel phylogenetic encoding of copy number (CN) data, the sitka transformation, that simplifies the site dependencies induced by rearrangements while still forming a sound foundation to phylogenetic inference. The sitka transformation allows us to design novel scalable Markov chain Monte Carlo (MCMC) algorithms. Moreover, we introduce a novel point mutation calling method that incorporates the CN data and the underlying phylogenetic tree to overcome the low per-cell coverage of scWGS. We demonstrate our method on three single cell datasets, including a novel PDX series, and analyse the topological properties of the inferred trees. Sitka is freely available athttps://github.com/UBC-Stat-ML/sitkatree.git.

The UK Biobank project is a prospective cohort study with deep genetic and phenotypic data collected on approximately 500,000 individuals from across the United Kingdom, aged between 40 and 69 at recruitment. The open resource is unique in its size and scope. A rich variety of phenotypic and health-related information is available on each participant, including biological measurements, lifestyle indicators, biomarkers in blood and urine, and imaging of the body and brain. Follow-up information is provided by linking health and medical records. Genome-wide genotype data have been collected on all participants, providing many opportunities for the discovery of new genetic associations and the genetic bases of complex traits. Here we describe the centralized analysis of the genetic data, including genotype quality, properties of population structure and relatedness of the genetic data, and efficient phasing and genotype imputation that increases the number of testable variants to around 96 million. Classical allelic variation at 11 human leukocyte antigen genes was imputed, resulting in the recovery of signals with known associations between human leukocyte antigen alleles and many diseases., Deep phenotype and genome-wide genetic data from 500,000 individuals from the UK Biobank, describing population structure and relatedness in the cohort, and imputation to increase the number of testable variants to 96 million.

Tagging and sequencing RNAs that are in close spatial proximity in the nucleus shows how the transcriptome is partitioned.

Nanopore DNA sequencing is limited by low base calling accuracy. Improved base-calling accuracy has so far relied on specialized base-calling algorithms, different nanopores and motor enzymes, or biochemical methods to re-read DNA molecules. Two primary error modes hamper sequencing accuracy: enzyme mis-steps and sequences with indistinguishable signals. We vary the driving voltage across an MspA nanopore between 100 to 200 mV with a frequency of 200 Hz, changing how the DNA strand moves through the nanopore. As a DNA helicase moves the DNA through the nanopore in discrete steps, the variable voltage positions the DNA continuously between these steps. The resulting electronic signal can be analysed to overcome the primary error modes in base-calling. Single-passage de novo base-calling accuracy in our device increases from 62.7 ± 0.5% with a constant driving voltage to 79.3 ± 0.3% with a variable driving voltage. Our variable-voltage sequencing mode is complementary to other advances in nanopore sequencing and is amenable to incorporation into other enzyme-actuated nanopore sequencing devices., Editor’s summary The accuracy of nanopore DNA sequencing is substantially improved by application of a variable-voltage sequencing mode.

Cellular engineering that allows budding yeast to survive with the four core human histones opens the door to exploring the function of histone variants and their modifications.

Ultradeep sequencing of Hi-C data from mouse cells in different developmental stages can address outstanding questions about 3D chromatin function.

Thin slices through nuclei provide an unbiased view of the complex 3D organization of mammalian genomes.

RNA-based toggle switches designed from first principles show high dynamic range and orthogonality.

CRISPR-mediated chromatin looping provides insight into the effect of genomic structure on gene expression.

Combining an inducible methyltransferase with a methylation-sensitive zinc finger creates an epigenetic memory circuit.

A CRISPR–Cas9-based approach identifies synonymous codons that can be reassigned to encode new chemical building blocks.

Sequential rounds of immunoprecipitation and barcoding reveal the genome-wide occurrence of paired histone marks.

By capturing and barcoding RNA in its native tissue location, researchers can visualize and quantify gene expression in situ.

It is believed that the introns are removed from pre-mRNAs during transcription, while the pre-mRNA is still tethered to the gene locus via RNA polymerase. However, during alternative splicing it is important that splicing be deferred until all of the exons and introns involved in the choice have been synthesized. We have developed an in situ RNA imaging method with single-molecule sensitivity to define the intracellular sites of splicing. Using this approach, we found that the normally tight coupling between transcription and splicing is broken in situations where the intron’s polypyrimidine tract is sequestered within strong secondary structures. We also found that in two cases of alternative splicing, in which certain exons are skipped due to the activity of the RNA binding proteins Sxl and PTB, splicing is uncoupled from transcription. This uncoupling occurs only on the perturbed introns, while the preceding and succeeding introns are removed co-transcriptionally.

An engineered dCas9–guide RNA complex containing three different transcriptional activation domains achieves consistently high expression of endogenous genes in a genome-wide screen.

5-hydroxymethylcytosine (5hmC) is a modified base present at low levels in diverse cell types in mammals1–5. 5hmC is generated by the TET family of Fe(II) and 2-oxoglutarate-dependent enzymes through oxidation of 5-methylcytosine (5mC)1,2,4–7. 5hmC and TET proteins have been implicated in stem cell biology and cancer1,4,5,8,9, but information on the genome-wide distribution of 5hmC is limited. Here we describe two novel and specific approaches to profile the genomic localization of 5hmC. The first approach, termed GLIB (glucosylation, periodate oxidation, biotinylation) uses a combination of enzymatic and chemical steps to isolate DNA fragments containing as few as a single 5hmC. The second approach involves conversion of 5hmC to cytosine 5-methylenesulphonate (CMS) by treatment of genomic DNA with sodium bisulphite, followed by immunoprecipitation of CMS-containing DNA with a specific antiserum to CMS5. High-throughput sequencing of 5hmC-containing DNA from mouse embryonic stem (ES) cells showed strong enrichment within exons and near transcriptional start sites. 5hmC was especially enriched at the start sites of genes whose promoters bear dual histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 4 trimethylation (H3K4me3) marks. Our results indicate that 5hmC has a probable role in transcriptional regulation, and suggest a model in which 5hmC contributes to the ‘poised’ chromatin signature found at developmentally-regulated genes in ES cells.

While the long non-coding RNAs (ncRNAs) constitute a large portion of the mammalian transcriptome, their biological functions has remained elusive. A few long ncRNAs that have been studied in any detail silence gene expression in processes such as X-inactivation and imprinting. We used a GENCODE annotation of the human genome to characterize over a thousand long ncRNAs that are expressed in multiple cell lines. Unexpectedly, we found an enhancer-like function for a set of these long ncRNAs in human cell lines. Depletion of a number of ncRNAs led to increased expression of their neighboring protein-coding genes, including the master regulator of hematopoiesis, SCL (also called TAL1), Snai1 and Snai2. Using heterologous transcription assays we demonstrated a requirement for the ncRNAs in mediating such enhancement of gene expression. These results reveal an unanticipated role for a class of long ncRNAs in activation of critical regulators of development and differentiation.

Short single-stranded DNA donors that asymmetrically span the Cas9 cut site show high efficiency in homology-directed editing.

Researchers show that to reliably establish protein interaction networks from microarray data it is necessary to measure saturation binding curves for each arrayed protein and its potential binding partners.

Dissecting the effect of a host's genetic background on circuit performance will allow better design.

With the help of engineered polymerases and reverse transcriptases, synthetic nucleic acids can encode and pass on genetic information.

Motivation: Nanopore sequencing may be the next disruptive technology in genomics, owing to its ability to detect single DNA molecules without prior amplification, lack of reliance on expensive optical components, and the ability to sequence long fragments. The MinION™ from Oxford Nanopore Technologies (ONT) is the first nanopore sequencer to be commercialized and is now available to early-access users. The MinION™ is a USB-connected, portable nanopore sequencer that permits real-time analysis of streaming event data. Currently, the research community lacks a standardized toolkit for the analysis of nanopore datasets. Results: We introduce poretools, a flexible toolkit for exploring datasets generated by nanopore sequencing devices from MinION™ for the purposes of quality control and downstream analysis. Poretools operates directly on the native FAST5 (an application of the HDF5 standard) file format produced by ONT and provides a wealth of format conversion utilities and data exploration and visualization tools. Availability and implementation: Poretools is an open-source software and is written in Python as both a suite of command line utilities and a Python application programming interface. Source code is freely available in Github at https://www.github.com/arq5x/poretools Contact: n.j.loman@bham.ac.uk and aaronquinlan@gmail.com Supplementary information: An IPython notebook demonstrating the functionality of poretools is in Github. Complete documentation is available at http://poretools.readthedocs.org.

By combining sequence motifs with biological information about kinases and phosphoproteins, researchers develop an algorithm, NetworKIN, to predict in vivo phosphorylation networks.

Capturing the interaction of promoters with their regulatory elements allows a high-resolution view into the regulatory landscape of chromatin.

With a new strategy for global metabolite profiling that identifies all substrates for a given enzyme comes a link between proteome and metabolome.

Binning based on differential read coverage, rather than sequence composition, allows separation of metagenomic sequence reads into species-level clusters that can be assembled into single chromosomes.

Tools to manipulate murine genes on a genome-wide scale and to phenotype their effects in animals are maturing.

The comprehensive mapping of transcripts, histone modifications and transcription factor binding allows for the functional annotation of fly and worm genomes.

The integration of quantitative proteomics and analysis by machine learning yields a refined list of proteins involved in chromosome function.

Researchers synthesized the first functional synthetic bacterial genome; repeating this feat with different bacteria will require further methodological development.

A genome-wide map of nucleosome positions in Schizosaccharomyces pombe points to a positioning mechanism different from that of other organisms.

Will some single molecule sequencing strategies be able to deliver on the promise of direct methyl cytosine sequencing?

Two methods enable the drawing of genome-wide chromatin interaction maps: one looks at protein-independent folding principles, the other at protein-mediated functional interactions.

With a modified polymerase and optimized oligonucleotide chemistry, Helicos' single-molecule sequencer takes on RNA.

To scale up the production and complexity of DNA nanostructures, researchers enlist the help of Escherichia coli to replicate and assemble them in vivo.