Showing papers by "Kevin L. Gunderson published in 2015"

Multiplex single cell profiling of chromatin accessibility by combinatorial cellular indexing.

Abstract: Technical advances have enabled the collection of genome and transcriptome data sets with single-cell resolution. However, single-cell characterization of the epigenome has remained challenging. Furthermore, because cells must be physically separated before biochemical processing, conventional single-cell preparatory methods scale linearly. We applied combinatorial cellular indexing to measure chromatin accessibility in thousands of single cells per assay, circumventing the need for compartmentalization of individual cells. We report chromatin accessibility profiles from more than 15,000 single cells and use these data to cluster cells on the basis of chromatin accessibility landscapes. We identify modules of coordinately regulated chromatin accessibility at the level of single cells both between and within cell types, with a scalable method that may accelerate progress toward a human cell atlas.

Subangstrom single-molecule measurements of motor proteins using a nanopore.

Abstract: Techniques for measuring the motion of single motor proteins, such as FRET and optical tweezers, are limited to a resolution of ∼300 pm. We use ion current modulation through the protein nanopore MspA to observe translocation of helicase Hel308 on DNA with up to ∼40 pm sensitivity. This approach should be applicable to any protein that translocates on DNA or RNA, including helicases, polymerases, recombinases and DNA repair enzymes.

Contiguity preserving transposition

Abstract: Embodiments provided herein relate to methods and compositions for preparing an immobilized library of barcoded DNA fragments of a target nucleic acid, identifying genomic variants, determining the contiguity information, phasing information, and methylation status of the target nucleic acid.

Biochemically activated electronic device

Abstract: A method of nucleic acid sequencing. The method can include the steps of (a) providing a polymerase tethered to a solid support charge sensor; (b) providing one or more nucleotides, whereby the presence of the nucleotide can be detected by the charge sensor; and (c) detecting incorporation of the nucleotide into a nascent strand complementary to a template nucleic acid.

Genetic and epigenetic profiling of CLL disease progression reveals limited somatic evolution and suggests a relationship to memory-cell development

Abstract: We examined genetic and epigenetic changes that occur during disease progression from indolent to aggressive forms of chronic lymphocytic leukemia (CLL) using serial samples from 27 patients. Analysis of DNA mutations grouped the leukemia cases into three categories: evolving (26%), expanding (26%) and static (47%). Thus, approximately three-quarters of the CLL cases had little to no genetic subclonal evolution. However, we identified significant recurrent DNA methylation changes during progression at 4752 CpGs enriched for regions near Polycomb 2 repressive complex (PRC2) targets. Progression-associated CpGs near the PRC2 targets undergo methylation changes in the same direction during disease progression as during normal development from naive to memory B cells. Our study shows that CLL progression does not typically occur via subclonal evolution, but that certain CpG sites undergo recurrent methylation changes. Our results suggest CLL progression may involve developmental processes shared in common with the generation of normal memory B cells.

Mutation in ST6GALNAC5 identified in family with coronary artery disease

Abstract: We aimed to identify the genetic cause of coronary artery disease (CAD) in an Iranian pedigree. Genetic linkage analysis identified three loci with an LOD score of 2.2. Twelve sequence variations identified by exome sequencing were tested for segregation with disease. A p.Val99Met causing mutation in ST6GALNAC5 was considered the likely cause of CAD. ST6GALNAC5 encodes sialyltransferase 7e. The variation affects a highly conserved amino acid, was absent in 800 controls, and was predicted to damage protein function. ST6GALNAC5 is positioned within loci previously linked to CAD-associated parameters. While hypercholesterolemia was a prominent feature in the family, clinical and genetic data suggest that this condition is not caused by the mutation in ST6GALNAC5. Sequencing of ST6GALNAC5 in 160 Iranian patients revealed a candidate causative stop-loss mutation in two other patients. The p.Val99Met and stop-loss mutations both caused increased sialyltransferase activity. Sequence data from combined Iranian and US controls and CAD affected individuals provided evidence consistent with potential role of ST6GALNAC5 in CAD. We conclude that ST6GALNAC5 mutations can cause CAD. There is substantial literature suggesting a relation between sialyltransferase and sialic acid levels and coronary disease. Our findings provide strong evidence for the existence of this relation.

Compositions, systems, and methods for detecting events using tethers anchored to or adjacent to nanopores

Abstract: Compositions, systems, and methods for detecting events are provided. A composition can include a nanopore including a first side, a second side, and an aperture extending through the first and second sides; and a permanent tether including head and tail regions and an elongated body disposed therebetween. The head region can be anchored to or adjacent to the first or second side of the nanopore. The elongated body including a reporter region can be movable within the aperture responsive to a first event occurring adjacent to the first side of the nanopore. For example, the reporter region is translationally movable toward the first side responsive to the first event, then toward the second side, then toward the first side responsive to a second event. The first event can include adding a first nucleotide to a polynucleotide. The second event can include adding a second nucleotide to the polynucleotide.

Transposase compositions for reduction of insertion bias

Abstract: Presented herein are methods and compositions for tagmentation of nucleic acids. The methods are useful for generating tagged DNA fragments that are qualitatively and quantitatively representative of the target nucleic acids in the sample from which they are generated.

Very long haplotype tracts characterized at high resolution from HLA homozygous cell lines.

Abstract: The HLA region of chromosome 6 contains the most polymorphic genes in humans. Spanning ~5 Mbp the densely packed region encompasses approximately 175 expressed genes including the highly polymorphic HLA class I and II loci. Most of the other genes and functional elements are also polymorphic, and many of them are directly implicated in immune function or immune-related disease. For these reasons, this complex genomic region is subject to intense scrutiny by researchers with the common goal of aiding further understanding and diagnoses of multiple immune-related diseases and syndromes. To aid assay development and characterization of the classical loci, a panel of cell lines partially or fully homozygous for HLA class I and II was assembled over time by the International Histocompatibility Working Group (IHWG). Containing a minimum of 88 unique HLA haplotypes, we show that this panel represents a significant proportion of European HLA allelic and haplotype diversity (60–95 %). Using a high-density whole genome array that includes 13,331 HLA region SNPs, we analyzed 99 IHWG cells to map the coordinates of the homozygous tracts at a fine scale. The mean homozygous tract length within chromosome 6 from these individuals is 21 Mbp. Within HLA, the mean haplotype length is 4.3 Mbp, and 65 % of the cell lines were shown to be homozygous throughout the entire region. In addition, four cell lines are homozygous throughout the complex KIR region of chromosome 19 (~250 kbp). The data we describe will provide a valuable resource for characterizing haplotypes, designing and refining imputation algorithms and developing assay controls.

Methods and arrays for producing and sequencing monoclonal clusters of nucleic acid

Abstract: Methods for capturing and amplifying target polynucleotides on a solid surface, in particular in a well in a microarray, wherein the microarray may comprise a) a substrate comprising at least one well, a surface surrounding the well and an inner well surface; b) a first layer covering the inner well surface and comprising at least one first capture primer pair; and c) a second layer covering the first layer and the surface surrounding the well Alternatively, the microarray may comprise a) a substrate comprising at least one well, a surface surrounding the well and an inner well surface; and b) a layer covering the inner well surface and comprising at least one first capture primer pair and at least one second capture primer pair In particular kinetic exclusion amplification is used in creating monoclonal populations of the nucleic acids in the wells The application also discloses a method for modifying an immobilized capture primer comprising: a) contacting a substrate comprising a plurality of immobilized capture primers with a plurality of template nucleic acids to produce one or more immobilized template nucleic acids,wherein the plurality of immobilized capture primers comprises a first plurality of primers comprising a 3'-terminal universal capture region Y, eg primer P5, and a second plurality of primers comprising a 3'-terminal universal capture region Z, eg primer P7; and wherein each template nucleic acid is flanked by a 5'-terminal and a 3'-terminal universal capture region Y or Z and comprises one or more, eg SapI, restriction sites and a target-specific capture region between the one or more restriction sites and the 3'-terminal universal capture region; and b) extending one or more immobilized capture primer Finally, the application discloses a method for modifying an immobilized capture primer comprising: a) contacting a substrate comprising a plurality of immobilized capture primers with a plurality of different seed nucleic acids to produce a plurality of different immobilized seed nucleic acids; b) extending two or more of the immobilized capture primers to produce a plurality of different immobilized extension products complementary to two or more of the plurality of different immobilized seed nucleic acids; and c) activating one immobilized extension product of the plurality of different immobilized extension products, to form an activated capture primer

Methods and compositions using one-sided transposition

Abstract: Embodiments provided herein relate to methods and compositions for next generation sequencing. Some embodiments include the preparation of a template library from a target nucleic acid using one-sided transposition, and sequencing the template library.

Compositions and methods for single molecular placement on a substrate

Abstract: Provided herein are methods and compositions for placing single target molecules on a patterned substrate.

Hybrid nanopore sensors

Abstract: In one aspect, a detection apparatus comprises a solid support with an array of solid-state nanopores and a plurality of lipid nanodiscs with protein nanopores, wherein the lipid nanodiscs are disposed on the solid support such that they form seals at the solid-state nanopores. In another aspect, a detection apparatus comprises one or more nanopores that are surrounded by a membrane, wherein each nanopore is tethered to an electrode.

Compositions, systems, and methods for detecting the presence of polymer subunits using chemiluminescence

Abstract: Under one aspect, a composition includes a substrate; a first polynucleotide coupled to the substrate; a second polynucleotide hybridized to the first polynucleotide; and a catalyst coupled to a first nucleotide of the second polynucleotide, the catalyst being operable to cause a chemiluminogenic molecule to emit a photon. Under another aspect, a method includes providing a catalyst operable to cause a first chemiluminogenic molecule to emit a photon; providing a substrate; providing a first polynucleotide coupled to the substrate; hybridizing a second polynucleotide to the first polynucleotide; coupling a first quencher to a first nucleotide of the second polynucleotide; and inhibiting, by the first quencher, photon emission by the first chemiluminogenic molecule.