Topics overview: Related analytes associated with physiology and disease, Contiguity preserving transposition” sequencing on beads, Inference of peptidoforms (IPF), Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site, A calcium- and light-gated switch to induce gene expression in activated neurons.
1. A wellness study of 108 individuals using personal, dense, dynamic data clouds
Personal data for 108 individuals were collected during a 9-month period, including whole genome sequences; clinical tests, metabolomes, proteomes, and microbiomes at three time points; and daily activity tracking. Using all of these data, Nathan D Price at Institute for Systems Biology in Washington, USA and his colleagues generated a correlation network that revealed communities of related analytes associated with physiology and disease. Connectivity within analyte communities enabled the identification of known and candidate biomarkers (e.g., gamma-glutamyltyrosine was densely interconnected with clinical analytes for cardiometabolic disease). They calculated polygenic scores from genome-wide association studies (GWAS) for 127 traits and diseases, and used these to discover molecular correlates of polygenic risk (e.g., genetic risk for inflammatory bowel disease was negatively correlated with plasma cystine). Finally, behavioral coaching informed by personal data helped participants to improve clinical biomarkers. Their results show that measurement of personal data clouds over time can improve our understanding of health and disease, including early transitions to disease states.
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2. Haplotype phasing of whole human genomes using bead-based barcode partitioning in a single tube
Haplotype-resolved genome sequencing promises to unlock a wealth of information in population and medical genetics. However, for the vast majority of genomes sequenced to date, haplotypes have not been determined because of cumbersome haplotyping workflows that require fractions of the genome to be sequenced in a large number of compartments. Here Fan Zhang at Advanced Research Department, Illumina in San Diego, California, USA and his colleagues demonstrate barcode partitioning of long DNA molecules in a single compartment using “on-bead” barcoded tagmentation. The key to the method that they call “contiguity preserving transposition” sequencing on beads (CPTv2-seq) is transposon-mediated transfer of homogenous populations of barcodes from beads to individual long DNA molecules that get fragmented at the same time (tagmentation). These are then processed to sequencing libraries wherein all sequencing reads originating from each long DNA molecule share a common barcode. Single-tube, bulk processing of long DNA molecules with ~150,000 different barcoded bead types provides a barcode-linked read structure that reveals long-range molecular contiguity. This technology provides a simple, rapid, plate-scalable and automatable route to accurate, haplotype-resolved sequencing, and phasing of structural variants of the genome.
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3. Inference and quantification of peptidoforms in large sample cohorts by SWATH-MS
Consistent detection and quantification of protein post-translational modifications (PTMs) across sample cohorts is a prerequisite for functional analysis of biological processes. Data-independent acquisition (DIA) is a bottom-up mass spectrometry approach that provides complete information on precursor and fragment ions. However, owing to the convoluted structure of DIA data sets, confident, systematic identification and quantification of peptidoforms has remained challenging. Here, George Rosenberger at Department of Biology, Institute of Molecular Systems Biology, ETH Zurich in Zurich, Switzerland and his colleagues present inference of peptidoforms (IPF), a fully automated algorithm that uses spectral libraries to query, validate and quantify peptidoforms in DIA data sets. The method was developed on data acquired by the DIA method SWATH-MS and benchmarked using a synthetic phosphopeptide reference data set and phosphopeptide-enriched samples. IPF reduced false site-localization by more than sevenfold compared with previous approaches, while recovering 85.4% of the true signals. Using IPF, they quantified peptidoforms in DIA data acquired from >200 samples of blood plasma of a human twin cohort and assessed the contribution of heritable, environmental and longitudinal effects on their PTMs.
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4. Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site
Many viral surface glycoproteins and cell surface receptors are homo-oligomers and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. Eva-Maria Strauch at Department of Biochemistry, University of Washington in Washington, USA and her colleagues describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites. In the first step, a small protein is designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. They use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.
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5. A calcium- and light-gated switch to induce gene expression in activated neurons.
Despite recent advances in optogenetics, it remains challenging to manipulate gene expression in specific populations of neurons. Dongmin Lee at Max Planck Florida Institute for Neuroscience in Florida, USA and her colleagues present a dual-protein switch system, Cal-Light, that translates neuronal-activity-mediated calcium signaling into gene expression in a light-dependent manner. In cultured neurons and brain slices, they show that Cal-Light drives expression of the reporter EGFP with high spatiotemporal resolution only in the presence of both blue light and calcium. Delivery of the Cal-Light components to the motor cortex of mice by viral vectors labels a subset of excitatory and inhibitory neurons related to learned lever-pressing behavior. By using Cal-Light to drive expression of the inhibitory receptor halorhodopsin (eNpHR), which responds to yellow light, they temporarily inhibit the lever-pressing behavior, confirming that the labeled neurons mediate the behavior. Thus, Cal-Light enables dissection of neural circuits underlying complex mammalian behaviors with high spatiotemporal precision.
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