Targeted short read sequencing and assembly of re-arrangements and candidate gene loci provide megabase diplotypes.

The human genome is composed of two haplotypes, otherwise called diplotypes, which denote phased polymorphisms and structural variations (SVs) that are derived from both parents. Diplotypes place genetic variants in the context of cis-related variants from a diploid genome. As a result, they provide valuable information about hereditary transmission, context of SV, regulation of gene expression and other features which are informative for understanding human genetics. Successful diplotyping with short read whole genome sequencing generally requires either a large population or parent-child trio samples. To overcome these limitations, we developed a targeted sequencing method for generating megabase (Mb)-scale haplotypes with short reads. One selects specific 0.1-0.2 Mb high molecular weight DNA targets with custom-designed Cas9-guide RNA complexes followed by sequencing with barcoded linked reads. To test this approach, we designed three assays, targeting the BRCA1 gene, the entire 4-Mb major histocompatibility complex locus and 18 well-characterized SVs, respectively. Using an integrated alignment- and assembly-based approach, we generated comprehensive variant diplotypes spanning the entirety of the targeted loci and characterized SVs with exact breakpoints. Our results were comparable in quality to long read sequencing.

Concentrating genomic length DNA in a microfabricated array.

We demonstrate that a microfabricated bump array can concentrate genomic-length DNA molecules efficiently at continuous, high flow velocities, up to 40 µm/s, if the single-molecule DNA globule has a sufficiently large shear modulus. Increase in the shear modulus is accomplished by compacting the DNA molecules to minimal coil size using polyethylene glycol (PEG) derived depletion forces. We map out the sweet spot, where concentration occurs, as a function of PEG concentration and flow speed using a combination of theoretical analysis and experiment. Purification of DNA from enzymatic reactions for next-generation DNA-sequencing libraries will be an important application of this development.

Targeted phasing of 2-200 kilobase DNA fragments with a short-read sequencer and a single-tube linked-read library method.

In the human genome, heterozygous sites refer to genomic positions with a different allele or nucleotide variant on the maternal and paternal chromosomes. Resolving these allelic differences by chromosomal copy, also known as phasing, is achievable on a short-read sequencer when using a library preparation method that captures long-range genomic information. TELL-Seq is a library preparation that captures long-range genomic information with the aid of molecular identifiers (barcodes). The same barcode is used to tag the reads derived from the same long DNA fragment within a range of up to 200 kilobases (kb), generating linked-reads. This strategy can be used to phase an entire genome. Here, we introduce a TELL-Seq protocol developed for targeted applications, enabling the phasing of enriched loci of varying sizes, purity levels, and heterozygosity. To validate this protocol, we phased 2-200 kb loci enriched with different methods: CRISPR/Cas9-mediated excision coupled with pulse-field electrophoresis for the longest fragments, CRISPR/Cas9-mediated protection from exonuclease digestion for mid-size fragments, and long PCR for the shortest fragments. All selected loci have known clinical relevance: BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, PMS2, SCN5A-SCN10A, and PKI3CA. Collectively, the analyses show that TELL-Seq can accurately phase 2-200 kb targets using a short-read sequencer.

A giant outburst two years before the core-collapse of a massive star.

The death of massive stars produces a variety of supernovae, which are linked to the structure of the exploding stars. The detection of several precursor stars of type II supernovae has been reported (see, for example, ref. 3), but we do not yet have direct information on the progenitors of the hydrogen-deficient type Ib and Ic supernovae. Here we report that the peculiar type Ib supernova SN 2006jc is spatially coincident with a bright optical transient that occurred in 2004. Spectroscopic and photometric monitoring of the supernova leads us to suggest that the progenitor was a carbon-oxygen Wolf-Rayet star embedded within a helium-rich circumstellar medium. There are different possible explanations for this pre-explosion transient. It appears similar to the giant outbursts of luminous blue variable stars (LBVs) of 60-100 solar masses, but the progenitor of SN 2006jc was helium- and hydrogen-deficient (unlike LBVs). An LBV-like outburst of a Wolf-Rayet star could be invoked, but this would be the first observational evidence of such a phenomenon. Alternatively, a massive binary system composed of an LBV that erupted in 2004, and a Wolf-Rayet star exploding as SN 2006jc, could explain the observations.

Targeted Phasing of 2-200 Kilobase DNA Fragments with a Short-Read Sequencer and a Single-Tube Linked-Read Library Method.

In the human genome, heterozygous sites are genomic positions with different alleles inherited from each parent. On average, there is a heterozygous site every 1-2 kilobases (kb). Resolving whether two alleles in neighboring heterozygous positions are physically linked-that is, phased-is possible with a short-read sequencer if the sequencing library captures long-range information. TELL-Seq is a library preparation method based on millions of barcoded micro-sized beads that enables instrument-free phasing of a whole human genome in a single PCR tube. TELL-Seq incorporates a unique molecular identifier (barcode) to the short reads generated from the same high-molecular-weight (HMW) DNA fragment (known as 'linked-reads'). However, genome-scale TELL-Seq is not cost-effective for applications focusing on a single locus or a few loci. Here, we present an optimized TELL-Seq protocol that enables the cost-effective phasing of enriched loci (targets) of varying sizes, purity levels, and heterozygosity. Targeted TELL-Seq maximizes linked-read efficiency and library yield while minimizing input requirements, fragment collisions on microbeads, and sequencing burden. To validate the targeted protocol, we phased seven 180-200 kb loci enriched by CRISPR/Cas9-mediated excision coupled with pulse-field electrophoresis, four 20 kb loci enriched by CRISPR/Cas9-mediated protection from exonuclease digestion, and six 2-13 kb loci amplified by PCR. The selected targets have clinical and research relevance (BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, PMS2, SCN5A-SCN10A, and PKI3CA). These analyses reveal that targeted TELL-Seq provides a reliable way of phasing allelic variants within targets (2-200 kb in length) with the low cost and high accuracy of short-read sequencing.

A fiber-optic DNA biosensor microarray for the analysis of gene expression.

A fiber-optic biosensor array is described for the simultaneous analysis of multiple DNA sequences. A bundle of optical fibers was assembled with each fiber carrying a different oligonucleotide probe immobilized on its distal end. Hybridization of fluorescently labeled complementary oligonucleotides to the array was monitored by observing the increase in fluorescence that accompanied binding. The approach enables fast (< 10 min) and sensitive (10 nM) detection to multiple DNA sequences simultaneously, with the potential for quantitative hybridization analysis.

The Source and Evolutionary History of a Microbial Contaminant Identified Through Soil Metagenomic Analysis.

In this study, strain-resolved metagenomics was used to solve a mystery. A 6.4-Mbp complete closed genome was recovered from a soil metagenome and found to be astonishingly similar to that of Delftia acidovorans SPH-1, which was isolated in Germany a decade ago. It was suspected that this organism was not native to the soil sample because it lacked the diversity that is characteristic of other soil organisms; this suspicion was confirmed when PCR testing failed to detect the bacterium in the original soil samples. D. acidovorans was also identified in 16 previously published metagenomes from multiple environments, but detailed-scale single nucleotide polymorphism analysis grouped these into five distinct clades. All of the strains indicated as contaminants fell into one clade. Fragment length anomalies were identified in paired reads mapping to the contaminant clade genotypes only. This finding was used to establish that the DNA was present in specific size selection reagents used during sequencing. Ultimately, the source of the contaminant was identified as bacterial biofilms growing in tubing. On the basis of direct measurement of the rate of fixation of mutations across the period of time in which contamination was occurring, we estimated the time of separation of the contaminant strain from the genomically sequenced ancestral population within a factor of 2. This research serves as a case study of high-resolution microbial forensics and strain tracking accomplished through metagenomics-based comparative genomics. The specific case reported here is unusual in that the study was conducted in the background of a soil metagenome and the conclusions were confirmed by independent methods.IMPORTANCE It is often important to determine the source of a microbial strain. Examples include tracking a bacterium linked to a disease epidemic, contaminating the food supply, or used in bioterrorism. Strain identification and tracking are generally approached by using cultivation-based or relatively nonspecific gene fingerprinting methods. Genomic methods have the ability to distinguish strains, but this approach typically has been restricted to isolates or relatively low-complexity communities. We demonstrate that strain-resolved metagenomics can be applied to extremely complex soil samples. We genotypically defined a soil-associated bacterium and identified it as a contaminant. By linking together snapshots of the bacterial genome over time, it was possible to estimate how long the contaminant had been diverging from a likely source population. The results are congruent with the derivation of the bacterium from a strain isolated in Germany and sequenced a decade ago and highlight the utility of metagenomics in strain tracking.

Structure of plectonemically supercoiled DNA.

Using electron microscopy and topological methods, we have deduced an average structure for negatively supercoiled circular DNA in solution. Our data suggest that DNA has a branched plectonemic (interwound) form over the range of supercoiling tested. The length of the superhelix axis is constant at 41% of the DNA length, whereas the superhelix radius decreases essentially hyperbolically as supercoiling increases. The number of supercoils is 89% of the linking deficit. Both writhe and twist change with supercoiling, but the ratio of the change in writhe to the change in twist is fixed at 2.6:1. The extent of branching of the superhelix axis is proportional to the length of the plasmid, but is insensitive to superhelix density. The relationship between DNA flexibility constants for twisting and bending calculated using our structural data is similar to that deduced from previous studies. The extended thin form of plectonemically supercoiled DNA offers little compaction for cellular packaging, but promotes interaction between cis-acting sequence elements that may be distant in primary structure. We discuss additional biological implications of our structural data.

Mutation typing using electrophoresis and gel-immobilized Acrydite probes.

A new electrophoresis technology for hybridization-based sequence detection and mutation typing is described. Intrinsic to this approach is copolymerization of specially modified oligonucleotide probes directly into polyacrylamide gels. Electrophoresis of single-stranded samples through gels containing specific immobilized probes results in hybridization-mediated capture of complementary targets. By increasing gel temperature or including denaturants in the buffer, the method can be used to type single-nucleotide polymorphisms. The method can easily be adapted to type mutations in PCR-amplified samples. Acrydite gel technology will also be useful for many other applications, including hybridization-based diagnostics, analysis of gene expression and purification of nucleic acids from biological samples.

Universal computer interface for the Cary 118C spectrophotometer.

We have constructed a simple interface which allows a computer to collect spectral data from and control the wavelength setting of a Cary 118C spectrophotometer. The interface is compatible with most types of computers; the computer need not be located near the spectrophotometer. Programs to operate the spectrophotometer can be written in high-level languages such as fortran or basic.

Forensic DNA testing on skeletal remains from mass graves: a pilot project in Guatemala.

A reliable method for extracting DNA from teeth was developed and successfully applied to a set of 12 skeletons recovered from two 10-year-old Guatemalan mass graves. Attempts to identify the remains by mitochondrial DNA (mtDNA) testing were hampered by low sequence diversity. Our findings demonstrate the feasibility of using DNA typing to identify victims from mass graves.

Plasmid recombination by the RecBCD pathway of Escherichia coli.

Previously, we demonstrated that exonuclease I-deficient strains of Escherichia coli accumulate high-molecular-weight linear plasmid concatemers when transformed with plasmids carrying the chi sequence (5'- GCTGGTGG-3') (M. M. Zaman and T. C. Boles, J. Bacteriol. 176:5093-5100, 1994). Since high-molecular weight linear DNA is believed to be the natural substrate for RecBCD-mediated recombination during conjugation (A. J. Clark and K. B. Low, p. 155-215, in K. B. Low, ed., The Recombination of Genetic Material, 1988), we analyzed the recombination frequencies of chi+ and chi0 plasmids in sbcB strains. Here, we report that chi sites stimulate plasmid recombination frequency by 16-fold in sbcB strains. Chi-stimulated plasmid recombination is dependent on RecBCD but is independent of RecF pathway genes. The distribution of recombination products suggests that high-molecular-weight linear plasmid DNA is a substrate for RecBCD-mediated recombination. Surprisingly, our data also suggest that chi+ plasmids also recombine by the RecBCD pathway in rec+ sbcB+ cells.

Chi-dependent formation of linear plasmid DNA in exonuclease-deficient recBCD+ strains of Escherichia coli.

Escherichia coli strains carrying mutations in sbcB (exonuclease I) or xthA (exonuclease III) accumulate high-molecular-weight linear plasmid concatemers when transformed with plasmids containing the chi sequence, 5'-GCTGGTGG-3'. Chi-dependent formation of high-molecular-weight plasmid DNA is dependent on recA and recF functions. In addition, chi stimulation occurs only in cis. Our data are consistent with models in which RecA and RecF proteins bind to and protect the DNA ends produced by RecBCD-chi interaction.

High-resolution mapping of carcinogen binding sites on DNA.

We have used a photochemical method to map covalent binding sites of the carcinogen benzo[a]pyrenediol epoxide (BPDE) within DNA from the transcriptional control region of the chicken adult beta-globin gene. Our preliminary low-resolution mapping has demonstrated that this region contains highly preferred BPDE binding sites [Boles, T.C., & Hogan, M.E. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5623-5627]. Here, we find that BPDE binding at individual G residues in this region is influenced by nearest-neighbor interactions and also by longer range interactions that may be attributable to sequence-specific variation of DNA secondary structure. Our findings suggest that long poly(dG) sequences should be preferred sites for BPDE action in other genes.

DNA structure equilibria in the human c-myc gene.

We have employed analytical S1 nuclease analysis to identify sites with altered DNA secondary structure in the human c-myc gene. We have mapped several sites of that kind in vitro at one-base resolution but have focused our attention on one particularly stable conformational isomer which occurs approximately 270 base pairs upstream from the preferred transcription origin. We have analyzed the kinetics of that conformational equilibrium as a function of supercoil density and enzyme concentration and find that DNA structure in this region is adequately modeled as a two-state equilibrium between an undistorted (S1 nuclease insensitive) and a distorted (S1-sensitive) state. We find that at fixed supercoil density, S1 nuclease cleavage at this DNA segment can be altered in vitro by a DNA sequence change as far away as 1500 bases. We also find that the S1 nuclease cleavage at this site can be dramatically enhanced by the binding of small RNA molecules. On the basis of an analysis of S1 cutting kinetics and an analysis of DNA sequence at the S1 cleavage site, we conclude that RNA may bind directly to DNA, thereby shifting the underlying conformational equilibrium. Together, these data suggest that as a class, short RNA molecules could serve as site-specific regulatory elements in the myc gene and elsewhere.

Site-specific carcinogen binding to DNA.

Benzo[alpha]pyrene diol epoxide (BPDE) is a well-studied environmental carcinogen that binds covalently to DNA. Here we describe a photochemical technique that allows us to map BPDE-binding sites within cloned gene sequences. The technique is based upon our observation that, when irradiated with laser light at 355 nm, one single-strand DNA cut is produced at each BPDE binding site. In initial experiments we have studied the distribution of such cuts in cloned DNA from the chicken adult beta-globin gene. We find that BPDE binding in this gene sequence is distinctly nonrandom. While several prominent BPDE-binding sites are evident, a 300-base-pair sequence immediately 5' to the RNA cap site is most strongly attacked by the carcinogen. This region is believed to contain important transcriptional control sequences. We discuss the possibility that sequence-specific binding to such regulatory elements may be an important feature of the mechanism of the carcinogen.

The helical repeat of underwound DNA in solution.

Closed circular DNA was relaxed with a topoisomerase in the presence of varying concentrations of the intercalating dye, ethidium bromide, to create underwound, planar DNA rings. We directly determined the helical repeat of these DNA molecules by the Gaussian center method and found that it varied as a simple predicted function of the degree of underwinding and the helical repeat of relaxed, dye-free DNA. We discuss these results in light of a recent mathematical treatment of DNA structure which predicts that the helical repeat of supercoiled DNA molecules in solution obeys the same function.

Analysis of the structure of dimeric DNA catenanes by electron microscopy.

We analyzed the structure of open-circular and supercoiled dimeric DNA catenanes generated by site-specific recombination in vitro. Electron microscopy of open-circular catenanes shows that the number of duplex crossings in a plane is a linear function of the number of catenane interlinks (Ca/2), and that the length of the catenane axis is constant, independent of Ca. These relationships are similar to those observed with supercoiled DNA. Statistical analyses reveal, however, that the conformations of the individual rings of the catenanes are similar to those of unlinked circles. The distribution of distances between randomly chosen points on separate rings depends strongly on Ca and is consistent with a sharp decrease in the center-of-mass separation between rings with increasing Ca. Singly linked supercoiled catenanes are seen by microscopy to be linked predominantly through terminal loops in the respective superhelices. The observations suggest that chain entropy is a major factor determining the conformation of DNA catenanes.

Immobilization of acrylamide-modified oligonucleotides by co-polymerization.

A flexible chemistry for solid phase attachment of oligonucleotides is described. Oligonucleotides bearing 5'-terminal acrylamide modifications efficiently co-polymerize with acrylamide monomers to form thermally stable DNA-containing polyacrylamide co-polymers. Co-polymerization attachment is specific for the terminal acrylamide group. Stable probe-containing layers are easily fabricated on supports bearing exposed acrylic groups, including plastic microtiter plates and silanized glass. Attachment can be accomplished using standard polyacrylamide gel recipes and polymerization techniques. Supports having a high surface density of hybridizable oligonucleotide (approximately 200 fmol/mm2) can be produced.