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 we demonstrate barcode partitioning of long DNA molecules in a single compartment using "on-bead" barcoded tagmentation.

Sequences of 95 human haplotypes reveal extreme coding variation in genes other than highly polymorphic and .

The most polymorphic part of the human genome, the encodes over 160 proteins of diverse function. Half of them, including the and genes, are directly involved in immune responses. Consequently, the region strongly associates with numerous diseases and clinical therapies.

Contiguity-Preserving Transposition Sequencing (CPT-Seq) for Genome-Wide Haplotyping, Assembly, and Single-Cell ATAC-Seq.

Most genomes to date have been sequenced without taking into account the diploid nature of the genome. However, the distribution of variants on each individual chromosome can (1) significantly impact gene regulation and protein function, (2) have important implications for analyses of population history and medical genetics, and (3) be of great value for accurate interpretation of medically relevant genetic variation. Here, we describe a comprehensive and detailed protocol for an ultra fast (<3 h library preparation), cost-effective, and scalable haplotyping method, named Contiguity Preserving Transposition sequencing or CPT-seq (Amini et al.

Massively multiplex single-cell Hi-C.

We present single-cell combinatorial indexed Hi-C (sciHi-C), a method that applies combinatorial cellular indexing to chromosome conformation capture. In this proof of concept, we generate and sequence six sciHi-C libraries comprising a total of 10,696 single cells. We use sciHi-C data to separate cells by karyotypic and cell-cycle state differences and identify cell-to-cell heterogeneity in mammalian chromosomal conformation.

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

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.

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

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.

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

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.

Mobile genetic elements and genome evolution 2014.

The Mobile Genetic Elements and Genome Evolution conference was hosted by Keystone Symposia in Santa Fe, NM USA, 9 March through 14 March 2014. The goal of this conference was to bring together scientists from around the world who study transposable elements in diverse organisms and researchers who study the impact these elements have on genome evolution. The meeting included over 200 scientists who participated through poster presentations, short talks selected from abstracts, and invited speakers.

In vitro, long-range sequence information for de novo genome assembly via transposase contiguity.

We describe a method that exploits contiguity preserving transposase sequencing (CPT-seq) to facilitate the scaffolding of de novo genome assemblies. CPT-seq is an entirely in vitro means of generating libraries comprised of 9216 indexed pools, each of which contains thousands of sparsely sequenced long fragments ranging from 5 kilobases to > 1 megabase. These pools are "subhaploid," in that the lengths of fragments contained in each pool sums to ∼5% to 10% of the full genome.

Haplotype-resolved whole-genome sequencing by contiguity-preserving transposition and combinatorial indexing.

Haplotype-resolved genome sequencing enables the accurate interpretation of medically relevant genetic variation, deep inferences regarding population history and non-invasive prediction of fetal genomes. We describe an approach for genome-wide haplotyping based on contiguity-preserving transposition (CPT-seq) and combinatorial indexing. Tn5 transposition is used to modify DNA with adaptor and index sequences while preserving contiguity.

Mutation in ST6GALNAC5 identified in family with coronary artery disease.

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.

Highly parallel oligonucleotide purification and functionalization using reversible chemistry.

We have developed a cost-effective, highly parallel method for purification and functionalization of 5'-labeled oligonucleotides. The approach is based on 5'-hexa-His phase tag purification, followed by exchange of the hexa-His tag for a functional group using reversible reaction chemistry. These methods are suitable for large-scale (micromole to millimole) production of oligonucleotides and are amenable to highly parallel processing of many oligonucleotides individually or in high complexity pools.

High density DNA methylation array with single CpG site resolution.

We have developed a new generation of genome-wide DNA methylation BeadChip which allows high-throughput methylation profiling of the human genome. The new high density BeadChip can assay over 480K CpG sites and analyze twelve samples in parallel. The innovative content includes coverage of 99% of RefSeq genes with multiple probes per gene, 96% of CpG islands from the UCSC database, CpG island shores and additional content selected from whole-genome bisulfite sequencing data and input from DNA methylation experts.

Genome-wide assessment of imprinted expression in human cells.

Background: Parent-of-origin-dependent expression of alleles, imprinting, has been suggested to impact a substantial proportion of mammalian genes. Its discovery requires allele-specific detection of expressed transcripts, but in some cases detected allelic expression bias has been interpreted as imprinting without demonstrating compatible transmission patterns and excluding heritable variation. Therefore, we utilized a genome-wide tool exploiting high density genotyping arrays in parallel measurements of genotypes in RNA and DNA to determine allelic expression across the transcriptome in lymphoblastoid cell lines (LCLs) and skin fibroblasts derived from families.

Deletion at fragile sites is a common and early event in Barrett's esophagus.

Barrett's esophagus (BE) is a premalignant intermediate to esophageal adenocarcinoma, which develops in the context of chronic inflammation and exposure to bile and acid. We asked whether there might be common genomic alterations that could be identified as potential clinical biomarker(s) for BE by whole genome profiling. We detected copy number alterations and/or loss of heterozygosity at 56 fragile sites in 20 patients with premalignant BE.

Computational analysis of whole-genome differential allelic expression data in human.

Allelic imbalance (AI) is a phenomenon where the two alleles of a given gene are expressed at different levels in a given cell, either because of epigenetic inactivation of one of the two alleles, or because of genetic variation in regulatory regions. Recently, Bing et al. have described the use of genotyping arrays to assay AI at a high resolution (approximately 750,000 SNPs across the autosomes).

Global patterns of cis variation in human cells revealed by high-density allelic expression analysis.

Cis-acting variants altering gene expression are a source of phenotypic differences. The cis-acting components of expression variation can be identified through the mapping of differences in allelic expression (AE), which is the measure of relative expression between two allelic transcripts. We generated a map of AE associated SNPs using quantitative measurements of AE on Illumina Human1M BeadChips.

Genome-wide DNA methylation profiling using Infinium® assay.

Aims: Bisulfite sequence analysis of individual CpG sites within genomic DNA is a powerful approach for methylation analysis in the genome. The major limitation of bisulfite-based methods is parallelization. Both array and next-generation sequencing technology are capable of addressing this bottleneck.

Whole-genome genotyping on bead arrays.

In this review, we describe the laboratory implementation of Infinium whole genome genotyping (WGG) technology for whole genome association studies and copy number studies. Briefly, the Infinium WGG assay employs a single tube whole genome amplification reaction to amplify the entire genome; genomic loci of interest are captured on an array by specific hybridization of picomolar concentrations amplified gDNA. After target capture, single nucleotide polymorphisms (SNPs) are genotyped on the array by a primer extension reaction using hapten-labeled nucleotides.

Design of tag SNP whole genome genotyping arrays.

Whole genome association studies have recently been enabled by combining tag SNP information derived from the International HapMap project with novel whole genome genotyping array technologies. In particular, Infinium whole genome genotyping (WGG) technology now has the power to genotype over 1 million SNPs on a single array. Additionally, this assay provides access to virtually any SNP in the genome enabling selection of optimized SNP content .

Single nucleotide polymorphism-based genome-wide chromosome copy change, loss of heterozygosity, and aneuploidy in Barrett's esophagus neoplastic progression.

Chromosome copy gain, loss, and loss of heterozygosity (LOH) involving most chromosomes have been reported in many cancers; however, less is known about chromosome instability in premalignant conditions. 17p LOH and DNA content abnormalities have been previously reported to predict progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA). Here, we evaluated genome-wide chromosomal instability in multiple stages of BE and EA in whole biopsies.

Increased LIS1 expression affects human and mouse brain development.

Deletions of the PAFAH1B1 gene (encoding LIS1) in 17p13.3 result in isolated lissencephaly sequence, and extended deletions including the YWHAE gene (encoding 14-3-3epsilon) cause Miller-Dieker syndrome. We identified seven unrelated individuals with submicroscopic duplication in 17p13.

Allele-specific gene expression patterns in primary leukemic cells reveal regulation of gene expression by CpG site methylation.

To identify genes that are regulated by cis-acting functional elements in acute lymphoblastic leukemia (ALL) we determined the allele-specific expression (ASE) levels of 2, 529 genes by genotyping a genome-wide panel of single nucleotide polymorphisms in RNA and DNA from bone marrow and blood samples of 197 children with ALL. Using a reproducible, quantitative genotyping method and stringent criteria for scoring ASE, we found that 16% of the analyzed genes display ASE in multiple ALL cell samples. For most of the genes, the level of ASE varied largely between the samples, from 1.

Delineation of the proximal 3q microdeletion syndrome.

Interstitial deletions of the proximal long arm of chromosome 3 are very rare and a defined clinical phenotype is not established yet. We report on the clinical, cytogenetic and molecular findings of a 20-month-old Hispanic male with a 2.5 Mb de novo deletion on q13.

Mosaic tetrasomy 12p with triplication of 12p detected by array-based comparative genomic hybridization of peripheral blood DNA.

A patient whose dysmorphism at birth was not diagnostic for Pallister-Killian syndrome (PKS) was found to have mosaic tetrasomy 12p by an array-based comparative genomic hybridization of peripheral blood DNA. He was determined to be mosaic for 46,XY,trp(12)(p11.2 --> p13) in cultured skin fibroblasts.