Development of coupling controlled polymerizations by adapter-ligation in mate-pair sequencing for detection of various genomic variants in one single assay.

The diversity of disease presentations warrants one single assay for detection and delineation of various genomic disorders. Herein, we describe a gel-free and biotin-capture-free mate-pair method through coupling Controlled Polymerizations by Adapter-Ligation (CP-AL). We first demonstrated the feasibility and ease-of-use in monitoring DNA nick translation and primer extension by limiting the nucleotide input. By coupling these two controlled polymerizations by a reported non-conventional adapter-ligation reaction 3' branch ligation, we evidenced that CP-AL significantly increased DNA circularization efficiency (by 4-fold) and was applicable for different sequencing methods but at a faction of current cost. Its advantages were further demonstrated by fully elimination of small-insert-contaminated (by 39.3-fold) with a ∼50% increment of physical coverage, and producing uniform genome/exome coverage and the lowest chimeric rate. It achieved single-nucleotide variants detection with sensitivity and specificity up to 97.3 and 99.7%, respectively, compared with data from small-insert libraries. In addition, this method can provide a comprehensive delineation of structural rearrangements, evidenced by a potential diagnosis in a patient with oligo-atheno-terato-spermia. Moreover, it enables accurate mutation identification by integration of genomic variants from different aberration types. Overall, it provides a potential single-integrated solution for detecting various genomic variants, facilitating a genetic diagnosis in human diseases.

Crystal Genetics, Inc.

Crystal Genetics, Inc. is an early-stage genetic test company, focused on achieving the highest possible clinical-grade accuracy and comprehensiveness for detecting germline (e.g., in hereditary cancer) and somatic (e.g., in early cancer detection) mutations. Crystal's mission is to significantly improve the health status of the population, by providing high accuracy, comprehensive, flexible and affordable genetic tests, primarily in cancer. Crystal's philosophy is that when it comes to detecting mutations that are strongly correlated with life-threatening diseases, the detection accuracy of every single mutation counts: a single false-positive error could cause severe anxiety for the patient. And, more importantly, a single false-negative error could potentially cost the patient's life. Crystal's objective is to eliminate both of these error types.

Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative, Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA.

Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital Sequencing™ is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient's cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing.

Detection and phasing of single base de novo mutations in biopsies from human in vitro fertilized embryos by advanced whole-genome sequencing.

Currently, the methods available for preimplantation genetic diagnosis (PGD) of in vitro fertilized (IVF) embryos do not detect de novo single-nucleotide and short indel mutations, which have been shown to cause a large fraction of genetic diseases. Detection of all these types of mutations requires whole-genome sequencing (WGS). In this study, advanced massively parallel WGS was performed on three 5- to 10-cell biopsies from two blastocyst-stage embryos. Both parents and paternal grandparents were also analyzed to allow for accurate measurements of false-positive and false-negative error rates. Overall, >95% of each genome was called. In the embryos, experimentally derived haplotypes and barcoded read data were used to detect and phase up to 82% of de novo single base mutations with a false-positive rate of about one error per Gb, resulting in fewer than 10 such errors per embryo. This represents a ∼ 100-fold lower error rate than previously published from 10 cells, and it is the first demonstration that advanced WGS can be used to accurately identify these de novo mutations in spite of the thousands of false-positive errors introduced by the extensive DNA amplification required for deep sequencing. Using haplotype information, we also demonstrate how small de novo deletions could be detected. These results suggest that phased WGS using barcoded DNA could be used in the future as part of the PGD process to maximize comprehensiveness in detecting disease-causing mutations and to reduce the incidence of genetic diseases.

Accurate whole-genome sequencing and haplotyping from 10 to 20 human cells.

Recent advances in whole-genome sequencing have brought the vision of personal genomics and genomic medicine closer to reality. However, current methods lack clinical accuracy and the ability to describe the context (haplotypes) in which genome variants co-occur in a cost-effective manner. Here we describe a low-cost DNA sequencing and haplotyping process, long fragment read (LFR) technology, which is similar to sequencing long single DNA molecules without cloning or separation of metaphase chromosomes. In this study, ten LFR libraries were made using only ∼100 picograms of human DNA per sample. Up to 97% of the heterozygous single nucleotide variants were assembled into long haplotype contigs. Removal of false positive single nucleotide variants not phased by multiple LFR haplotypes resulted in a final genome error rate of 1 in 10 megabases. Cost-effective and accurate genome sequencing and haplotyping from 10-20 human cells, as demonstrated here, will enable comprehensive genetic studies and diverse clinical applications.

Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays.

Genome sequencing of large numbers of individuals promises to advance the understanding, treatment, and prevention of human diseases, among other applications. We describe a genome sequencing platform that achieves efficient imaging and low reagent consumption with combinatorial probe anchor ligation chemistry to independently assay each base from patterned nanoarrays of self-assembling DNA nanoballs. We sequenced three human genomes with this platform, generating an average of 45- to 87-fold coverage per genome and identifying 3.2 to 4.5 million sequence variants per genome. Validation of one genome data set demonstrates a sequence accuracy of about 1 false variant per 100 kilobases. The high accuracy, affordable cost of $4400 for sequencing consumables, and scalability of this platform enable complete human genome sequencing for the detection of rare variants in large-scale genetic studies.

Retention of histidine-containing peptides on a nickel affinity column.

The retention of histidine-containing peptides in immobilized metal-affinity chromatography is studied using several hundred modeled peptides. Retention is driven primarily by the number of histidine residues; however, the amino acid composition in the immediate vicinity plays a significant role. Specifically, the arginine and tryptophan content has to be taken into consideration. During the course of this study, an alternative tag that can be used similarly to a polyhistidine tag is discovered.

A multiplexed protein kinase assay.

We report a novel protein kinase assay designed for high-throughput detection of one or many kinases in a complex mixture. A solution-phase phosphorylation reaction is performed on 900 different peptide substrates, each covalently linked to an oligonucleotide tag. After incubation, phosphoserine, phosphothreonine, and phosphotyrosine are chemically labeled, and the substrates are hybridized to a microarray with oligonucleotides complementary to the tags to read out the phosphorylation state of each peptide. Because protein kinases act on more than one peptide sequence, each kinase can be characterized by a unique signature of phosphorylation activity on multiple substrates. Using this method, we determined signatures for 26 purified kinases and demonstrated that enzyme mixtures can be screened for activity and selectivity of inhibition.

High-throughput SNP genotyping on universal bead arrays.

We have developed a flexible, accurate and highly multiplexed SNP genotyping assay for high-throughput genetic analysis of large populations on a bead array platform. The novel genotyping system combines high assay conversion rate and data quality with >1500 multiplexing, and Array of Arrays formats. Genotyping assay oligos corresponding to specific SNP sequences are each linked to a unique sequence (address) that can hybridize to its complementary strand on universal arrays. The arrays are made of beads located in microwells of optical fiber bundles (Sentrix Array Matrix) or silicon slides (Sentrix BeadChip). The optical fiber bundles are further organized into a matrix that matches a 96-well microtiter plate. The arrays on the silicon slides are multi-channel pipette compatible for loading multiple samples onto a single silicon slide. These formats allow many samples to be processed in parallel. This genotyping system enables investigators to generate approximately 300,000 genotypes per day with minimal equipment requirements and greater than 1.6 million genotypes per day in a robotics-assisted process. With a streamlined and comprehensive assay, this system brings a new level of flexibility, throughput, and affordability to genetic research.

Decoding randomly ordered DNA arrays.

We have developed a simple and efficient algorithm to identify each member of a large collection of DNA-linked objects through the use of hybridization, and have applied it to the manufacture of randomly assembled arrays of beads in wells. Once the algorithm has been used to determine the identity of each bead, the microarray can be used in a wide variety of applications, including single nucleotide polymorphism genotyping and gene expression profiling. The algorithm requires only a few labels and several sequential hybridizations to identify thousands of different DNA sequences with great accuracy. We have decoded tens of thousands of arrays, each with 1520 sequences represented at approximately 30-fold redundancy by up to approximately 50,000 beads, with a median error rate of <1 x 10(-4) per bead. The approach makes use of error checking codes and provides, for the first time, a direct functional quality control of every element of each array that is manufactured. The algorithm can be applied to any spatially fixed collection of objects or molecules that are associated with specific DNA sequences.

A highly informative SNP linkage panel for human genetic studies.

We have developed a highly informative set of single-nucleotide polymorphism (SNP) assays designed for linkage mapping of the human genome. These assays were developed on a robust multiplexed assay system to provide a combination of very high accuracy and data completeness with high throughput for linkage studies. The linkage panel is comprised of approximately 4,700 SNPs with 0.39 average minor allele frequency and 624-kb average spacing. Based on almost 2 million genotypes, data quality was shown to be extremely high, with a 99.94% call rate, >99.99% reproducibility and 99.995% genotypes consistent with mendelian inheritance. We constructed a genetic map with an average 1.5-cM resolution using series of 28 CEPH pedigrees. The relative information content of this panel was higher than those of commonly used STR marker panels. The potent combination of this SNP linkage panel with the multiplexed assay system provides a previously unattainable level of performance for linkage studies.