High-throughput single-cell DNA sequencing of acute myeloid leukemia tumors with droplet microfluidics.

To enable the characterization of genetic heterogeneity in tumor cell populations, we developed a novel microfluidic approach that barcodes amplified genomic DNA from thousands of individual cancer cells confined to droplets. The barcodes are then used to reassemble the genetic profiles of cells from next-generation sequencing data. By using this approach, we sequenced longitudinally collected acute myeloid leukemia (AML) tumor populations from two patients and genotyped up to 62 disease relevant loci across more than 16,000 individual cells. Targeted single-cell sequencing was able to sensitively identify cells harboring pathogenic mutations during complete remission and uncovered complex clonal evolution within AML tumors that was not observable with bulk sequencing. We anticipate that this approach will make feasible the routine analysis of AML heterogeneity, leading to improved stratification and therapy selection for the disease.

DNA sequencing versus standard prenatal aneuploidy screening.

BACKGROUND: In high-risk pregnant women, noninvasive prenatal testing with the use of massively parallel sequencing of maternal plasma cell-free DNA (cfDNA testing) accurately detects fetal autosomal aneuploidy. Its performance in low-risk women is unclear. METHODS: At 21 centers in the United States, we collected blood samples from women with singleton pregnancies who were undergoing standard aneuploidy screening (serum biochemical assays with or without nuchal translucency measurement). We performed massively parallel sequencing in a blinded fashion to determine the chromosome dosage for each sample. The primary end point was a comparison of the false positive rates of detection of fetal trisomies 21 and 18 with the use of standard screening and cfDNA testing. Birth outcomes or karyotypes were the reference standard. RESULTS: The primary series included 1914 women (mean age, 29.6 years) with an eligible sample, a singleton fetus without aneuploidy, results from cfDNA testing, and a risk classification based on standard screening. For trisomies 21 and 18, the false positive rates with cfDNA testing were significantly lower than those with standard screening (0.3% vs. 3.6% for trisomy 21, P<0.001; and 0.2% vs. 0.6% for trisomy 18, P=0.03). The use of cfDNA testing detected all cases of aneuploidy (5 for trisomy 21, 2 for trisomy 18, and 1 for trisomy 13; negative predictive value, 100% [95% confidence interval, 99.8 to 100]). The positive predictive values for cfDNA testing versus standard screening were 45.5% versus 4.2% for trisomy 21 and 40.0% versus 8.3% for trisomy 18. CONCLUSIONS: In a general obstetrical population, prenatal testing with the use of cfDNA had significantly lower false positive rates and higher positive predictive values for detection of trisomies 21 and 18 than standard screening. (Funded by Illumina; ClinicalTrials.gov number, NCT01663350.).

Synchrotron micro-scale measurement of metal distributions in Phragmites australis and Typha latifolia root tissue from an urban brownfield site.

Liberty State Park in New Jersey, USA, is a "brownfield" site containing various levels of contaminants. To investigate metal uptake and distributions in plants on the brownfield site, Phragmites australis and Typha latifolia were collected in Liberty State Park during the growing season (May-September) in 2011 at two sites with the high and low metal loads, respectively. The objective of this study was to understand the metal (Fe, Mn, Cu, Pb and Zn) concentration and spatial distributions in P. australis and T. latifolia root systems with micro-meter scale resolution using synchrotron X-ray microfluorescence (µXRF) and synchrotron X-ray computed microtomography (µCMT) techniques. The root structure measurement by synchrotron µCMT showed that high X-ray attenuation substance appeared in the epidermis. Synchrotron µXRF measurement showed that metal concentrations and distributions in the root cross-section between epidermis and vascular tissue were statistically different. Significant correlations were found between metals (Cu, Mn, Pb and Zn) and Fe in the epidermis, implying that metals were scavenged by Fe oxides. The results from this study suggest that the expression of metal transport and accumulation within the root systems may be element specific. The information derived from this study can improve our current knowledge of the wetland plant ecological function in brownfield remediation.

Synchrotron study of metal localization in Typha latifolia L. root sections.

Understanding mechanisms that control plant root metal assimilation in soil is critical to the sustainable management of metal-contaminated land. With the assistance of the synchrotron X-ray fluorescence technique, this study investigated possible mechanisms that control the localization of Fe, Cu, Mn, Pb and Zn in the root tissues of Typha latifolia L. collected from a contaminated wetland. Metal localizations especially in the case of Fe and Pb in the dermal tissue and the vascular bundles were different. Cluster analysis was performed to divide the dermal tissue into iron-plaque-enriched dermal tissue and regular dermal tissue based on the spatial distribution of Pb and Fe. Factor analysis showed that Cu and Zn were closely correlated to each other in the dermal tissues. The association of Cu, Zn and Mn with Fe was strong in both regular dermal tissue and iron-plaque-enriched dermal tissue, while significant (p < 0.05) correlation of Fe with Pb was only observed in tissues enriched with iron plaque. In the vascular bundles, Zn, Mn and Cu showed strong association, suggesting that the localization of these three elements was controlled by a similar mechanism. Iron plaque in the peripheral dermal tissues acted as a barrier for Pb and a buffer for Zn, Cu and Mn. The Casparian strip regulated the transportation of metals from dermal tissues to the vascular bundles. The results suggested that the mechanisms controlling metal localization in root tissues varied with both tissue types and metals.

Evaluating potential for whole-genome studies in Kosrae, an isolated population in Micronesia.

Whole-genome association studies are predicted to be especially powerful in isolated populations owing to increased linkage disequilibrium (LD) and decreased allelic diversity, but this possibility has not been empirically tested. We compared genome-wide data on 113,240 SNPs typed on 30 trios from the Pacific island of Kosrae to the same markers typed in the 270 samples from the International HapMap Project. The extent of LD is longer and haplotype diversity is lower in Kosrae than in the HapMap populations. More than 98% of Kosraen haplotypes are present in HapMap populations, indicating that HapMap will be useful for genetic studies on Kosrae. The long-range LD around common alleles and limited diversity result in improved efficiency in genetic studies in this population and augments the power to detect association of 'hidden SNPs'.

Population-genetic nature of copy number variations in the human genome.

Copy number variations (CNVs) are universal genetic variations, and their association with disease has been increasingly recognized. We designed high-density microarrays for CNVs, and detected 3000-4000 CNVs (4-6% of the genomic sequence) per population that included CNVs previously missed because of smaller sizes and residing in segmental duplications. The patterns of CNVs across individuals were surprisingly simple at the kilo-base scale, suggesting the applicability of a simple genetic analysis for these genetic loci. We utilized the probabilistic theory to determine integer copy numbers of CNVs and employed a recently developed phasing tool to estimate the population frequencies of integer copy number alleles and CNV-SNP haplotypes. The results showed a tendency toward a lower frequency of CNV alleles and that most of our CNVs were explained only by zero-, one- and two-copy alleles. Using the estimated population frequencies, we found several CNV regions with exceptionally high population differentiation. Investigation of CNV-SNP linkage disequilibrium (LD) for 500-900 bi- and multi-allelic CNVs per population revealed that previous conflicting reports on bi-allelic LD were unexpectedly consistent and explained by an LD increase correlated with deletion-allele frequencies. Typically, the bi-allelic LD was lower than SNP-SNP LD, whereas the multi-allelic LD was somewhat stronger than the bi-allelic LD. After further investigation of tag SNPs for CNVs, we conclude that the customary tagging strategy for disease association studies can be applicable for common deletion CNVs, but direct interrogation is needed for other types of CNVs.

Global variation in copy number in the human genome.

Copy number variation (CNV) of DNA sequences is functionally significant but has yet to be fully ascertained. We have constructed a first-generation CNV map of the human genome through the study of 270 individuals from four populations with ancestry in Europe, Africa or Asia (the HapMap collection). DNA from these individuals was screened for CNV using two complementary technologies: single-nucleotide polymorphism (SNP) genotyping arrays, and clone-based comparative genomic hybridization. A total of 1,447 copy number variable regions (CNVRs), which can encompass overlapping or adjacent gains or losses, covering 360 megabases (12% of the genome) were identified in these populations. These CNVRs contained hundreds of genes, disease loci, functional elements and segmental duplications. Notably, the CNVRs encompassed more nucleotide content per genome than SNPs, underscoring the importance of CNV in genetic diversity and evolution. The data obtained delineate linkage disequilibrium patterns for many CNVs, and reveal marked variation in copy number among populations. We also demonstrate the utility of this resource for genetic disease studies.

High throughput single-cell detection of multiplex CRISPR-edited gene modifications.

CRISPR-Cas9 gene editing has transformed our ability to rapidly interrogate the functional impact of somatic mutations in human cancers. Droplet-based technology enables the analysis of Cas9-introduced gene edits in thousands of single cells. Using this technology, we analyze Ba/F3 cells engineered to express single or multiplexed loss-of-function mutations recurrent in chronic lymphocytic leukemia. Our approach reliably quantifies mutational co-occurrences, zygosity status, and the occurrence of Cas9 edits at single-cell resolution.

Improved detection of global copy number variation using high density, non-polymorphic oligonucleotide probes.

BACKGROUND: DNA sequence diversity within the human genome may be more greatly affected by copy number variations (CNVs) than single nucleotide polymorphisms (SNPs). Although the importance of CNVs in genome wide association studies (GWAS) is becoming widely accepted, the optimal methods for identifying these variants are still under evaluation. We have previously reported a comprehensive view of CNVs in the HapMap DNA collection using high density 500 K EA (Early Access) SNP genotyping arrays which revealed greater than 1,000 CNVs ranging in size from 1 kb to over 3 Mb. Although the arrays used most commonly for GWAS predominantly interrogate SNPs, CNV identification and detection does not necessarily require the use of DNA probes centered on polymorphic nucleotides and may even be hindered by the dependence on a successful SNP genotyping assay. RESULTS: In this study, we have designed and evaluated a high density array predicated on the use of non-polymorphic oligonucleotide probes for CNV detection. This approach effectively uncouples copy number detection from SNP genotyping and thus has the potential to significantly improve probe coverage for genome-wide CNV identification. This array, in conjunction with PCR-based, complexity-reduced DNA target, queries over 1.3 M independent NspI restriction enzyme fragments in the 200 bp to 1100 bp size range, which is a several fold increase in marker density as compared to the 500 K EA array. In addition, a novel algorithm was developed and validated to extract CNV regions and boundaries. CONCLUSION: Using a well-characterized pair of DNA samples, close to 200 CNVs were identified, of which nearly 50% appear novel yet were independently validated using quantitative PCR. The results indicate that non-polymorphic probes provide a robust approach for CNV identification, and the increasing precision of CNV boundary delineation should allow a more complete analysis of their genomic organization.

High-density oligonucleotide array with sub-kilobase resolution reveals breakpoint information of submicroscopic deletions in nevoid basal cell carcinoma syndrome.

Small submicroscopic genomic deletions and duplications constitute up to 15% of all mutations underlying human monogenic diseases. In this study, we used newly designed high-resolution oligonucleotide microarrays with a median distance between the probes of 776 bp (average probe interval 2,271 bp) to detect gene deletions in nevoid basal cell carcinoma syndrome (NBCCS) patients. NBCCS, also called Gorlin syndrome, is characterized by developmental defects and tumorigenesis such as medulloblastomas and basal cell carcinomas, caused by mutations of the human patched-1 (PTCH1) gene. Two out of three deletions could not be detected by a conventional chromosomal analysis. A submicroscopic deletion as small as 165 kb was detected affecting only PTCH1, whereas the other two deletions were much larger (5 and 11 Mb). We demonstrated not only the exact number of genes involved in the deletion but also rapidly determined the junction sequences after pinpointing the breakpoint regions in all individuals analyzed. This report of an array-based determination of junction sequences of long deletions circumvented a labor-intensive analysis such as Southern blotting or FISH. Alu-mediated recombination in one case and non-homologous end joining in the other two were probably implicated in the generation of deletions. This method will contribute to the understanding of molecular pathogenesis of gene deletions as well as rapid genetic testing.

Large-scale genotyping of complex DNA.

Genetic studies aimed at understanding the molecular basis of complex human phenotypes require the genotyping of many thousands of single-nucleotide polymorphisms (SNPs) across large numbers of individuals. Public efforts have so far identified over two million common human SNPs; however, the scoring of these SNPs is labor-intensive and requires a substantial amount of automation. Here we describe a simple but effective approach, termed whole-genome sampling analysis (WGSA), for genotyping thousands of SNPs simultaneously in a complex DNA sample without locus-specific primers or automation. Our method amplifies highly reproducible fractions of the genome across multiple DNA samples and calls genotypes at >99% accuracy. We rapidly genotyped 14,548 SNPs in three different human populations and identified a subset of them with significant allele frequency differences between groups. We also determined the ancestral allele for 8,386 SNPs by genotyping chimpanzee and gorilla DNA. WGSA is highly scaleable and enables the creation of ultrahigh density SNP maps for use in genetic studies.

CARAT: a novel method for allelic detection of DNA copy number changes using high density oligonucleotide arrays.

BACKGROUND: DNA copy number alterations are one of the main characteristics of the cancer cell karyotype and can contribute to the complex phenotype of these cells. These alterations can lead to gains in cellular oncogenes as well as losses in tumor suppressor genes and can span small intervals as well as involve entire chromosomes. The ability to accurately detect these changes is central to understanding how they impact the biology of the cell. RESULTS: We describe a novel algorithm called CARAT (Copy Number Analysis with Regression And Tree) that uses probe intensity information to infer copy number in an allele-specific manner from high density DNA oligonuceotide arrays designed to genotype over 100,000 SNPs. Total and allele-specific copy number estimations using CARAT are independently evaluated for a subset of SNPs using quantitative PCR and allelic TaqMan reactions with several human breast cancer cell lines. The sensitivity and specificity of the algorithm are characterized using DNA samples containing differing numbers of X chromosomes as well as a test set of normal individuals. Results from the algorithm show a high degree of agreement with results from independent verification methods. CONCLUSION: Overall, CARAT automatically detects regions with copy number variations and assigns a significance score to each alteration as well as generating allele-specific output. When coupled with SNP genotype calls from the same array, CARAT provides additional detail into the structure of genome wide alterations that can contribute to allelic imbalance.

Large-scale SNP analysis reveals clustered and continuous patterns of human genetic variation.

Understanding the distribution of human genetic variation is an important foundation for research into the genetics of common diseases. Some of the alleles that modify common disease risk are themselves likely to be common and, thus, amenable to identification using gene-association methods. A problem with this approach is that the large sample sizes required for sufficient statistical power to detect alleles with moderate effect make gene-association studies susceptible to false-positive findings as the result of population stratification. Such type I errors can be eliminated by using either family-based association tests or methods that sufficiently adjust for population stratification. These methods require the availability of genetic markers that can detect and, thus, control for sources of genetic stratification among populations. In an effort to investigate population stratification and identify appropriate marker panels, we have analysed 11,555 single nucleotide polymorphisms in 203 individuals from 12 diverse human populations. Individuals in each population cluster to the exclusion of individuals from other populations using two clustering methods. Higher-order branching and clustering of the populations are consistent with the geographic origins of populations and with previously published genetic analyses. These data provide a valuable resource for the definition of marker panels to detect and control for population stratification in population-based gene identification studies. Using three US resident populations (European-American, African-American and Puerto Rican), we demonstrate how such studies can proceed, quantifying proportional ancestry levels and detecting significant admixture structure in each of these populations.

MARA: a novel approach for highly multiplexed locus-specific SNP genotyping using high-density DNA oligonucleotide arrays.

We have developed a locus-specific DNA target preparation method for highly multiplexed single nucleotide polymorphism (SNP) genotyping called MARA (Multiplexed Anchored Runoff Amplification). The approach uses a single primer per SNP in conjunction with restriction enzyme digested, adapter-ligated human genomic DNA. Each primer is composed of common sequence at the 5' end followed by locus-specific sequence at the 3' end. Following a primary reaction in which locus-specific products are generated, a secondary universal amplification is carried out using a generic primer pair corresponding to the oligonucleotide and genomic DNA adapter sequences. Allele discrimination is achieved by hybridization to high-density DNA oligonucleotide arrays. Initial multiplex reactions containing either 250 primers or 750 primers across nine DNA samples demonstrated an average sample call rate of approximately 95% for 250- and 750-plex MARA. We have also evaluated >1000- and 4000-primer plex MARA to genotype SNPs from human chromosome 21. We have identified a subset of SNPs corresponding to a primer conversion rate of approximately 75%, which show an average call rate over 95% and concordance >99% across seven DNA samples. Thus, MARA may potentially improve the throughput of SNP genotyping when coupled with allele discrimination on high-density arrays by allowing levels of multiplexing during target generation that far exceed the capacity of traditional multiplex PCR.

Synchrotron micro-scale study of trace metal transport and distribution in Spartina alterniflora root system in Yangtze River intertidal zone.

This study is focused on micro-scale measurement of metal (Ca, Cl, Fe, K, Mn, Cu, Pb, and Zn) distributions in Spartina alterniflora root system. The root samples were collected in the Yangtze River intertidal zone in July 2013. Synchrotron X-ray fluorescence (XRF), computed microtomography (CMT), and X-ray absorption near-edge structure (XANES) techniques, which provide micro-meter scale analytical resolution, were applied to this study. Although it was found that the metals of interest were distributed in both epidermis and vascular tissue with the varying concentrations, the results showed that Fe plaque was mainly distributed in the root epidermis. Other metals (e.g., Cu, Mn, Pb, and Zn) were correlated with Fe in the epidermis possibly due to scavenge by Fe plaque. Relatively high metal concentrations were observed in the root hair tip. This micro-scale investigation provides insights of understanding the metal uptake and spatial distribution as well as the function of Fe plaque governing metal transport in the root system.

Calcium compartments in brain.

Excellent progress has been made toward understanding the physiology and pharmacology of specific calcium-related cellular processes of the brain, but few studies have provided an integrated view of brain calcium kinetics. To further the knowledge of the size and binding properties of brain calcium compartments, the authors have conducted a series of experiments in hippocampal brain slices exposed to high and low extracellular calcium. Slices were incubated in buffers containing 0.001 to 4.5 mmol/L calcium for up to 75 minutes. Slice calcium content was analyzed by three methods: exchange equilibrium with 45Ca, synchrotron-radiation-induced x-ray emission, and inductively coupled plasma. Data were analyzed using a model based on a Langmuir isotherm for two independent sites, with additional extracellular and bound compartments. In parallel experiments, altered low calcium had no effect on slice histology and only mild effects on slice adenylates. When combined with prior 45Ca and fluorescent probe binding experiments, these results suggest that there are at least five kinetically distinct calcium compartments: (1) free extracellular (approximately 10%); (2) loosely associated extracellular plasma membrane (approximately 55%); (3) intracellular compartment with moderate avidity (approximately 17%); (4) tightly bound, nonexchangeable intracellular compartment ( approximately 15%); and (5) free cytoplasmic (<0.01%). If only the third compartment is considered a potential calcium buffer, then the buffering ratio is calculated to be approximately 2,700:1, but if the second compartment is also included, then the buffering ratio would be approximately 13,000:1. This may explain the wide range of estimates observed by fluorescent probe studies.

Synchrotron radiation-induced X-ray emission to identify metal ions in preparations of purified protein.

The suitability of synchrotron radiation-induced X-ray emission (SRIXE) for the detection and identification of metal ions in preparations of purified, soluble proteins was tested. Glutathione S-transferase fused to the proximal zinc finger motif of human transcription factor IIIA or to the cysteine-rich motif of poliovirus protein 2C was expressed in bacteria and purified by affinity chromatography. Aqueous samples containing the purified proteins were analyzed with SRIXE, and trace amounts of zinc and iron were detected. Mutation of the zinc-coordinating residues in the cysteine-rich motif of poliovirus protein 2C resulted in the loss of the zinc-binding ability. Relative quantities of metal in the protein preparations as determined by SRIXE corresponded well with the metal:protein ratios calculated by using a 4-(2-pyridylazo)resorcinol-based assay. We conclude that SRIXE is an accurate, sensitive, and simple method for the detection and identification of protein-bound metal ions in small amounts of sample. Thus, SRIXE may have wide use as a particularly effective method for rapidly determining trace metals in microarray samples.

The CRF(1) receptor antagonist DMP696 produces anxiolytic effects and inhibits the stress-induced hypothalamic-pituitary-adrenal axis activation without sedation or ataxia in rats.

RATIONALE: CRF(1) antagonists may be effective in the treatment of anxiety disorders while having fewer side effects compared with classical benzodiazepines. OBJECTIVES: The effects of a small molecule selective CRF(1) antagonist DMP696 on anxiety-like behaviors and stress-induced increases in corticosterone in rats exposed to a novel environment and on locomotor activity and motor coordination were determined in rats. These effects of DMP696 were compared with those produced by the classical benzodiazepine chlordiazepoxide (CDP). METHODS: DMP696 or CDP were administered PO, 60 minutes before behavioral testing in rats. Their effects on latency to exit a dark chamber and stress-induced increase in corticosterone in the Defensive Withdrawal test (an animal model of anxiety), locomotor activity, and rotorod performance (measure of ataxia) were determined. RESULTS: DMP696 significantly reduced exit latency and reversed the stress-induced increase in corticosterone in the Defensive Withdrawal test at doses of 3.0-10 mg/kg and higher. In contrast, CDP significantly decreased exit latency at 10 and 30 mg/kg, but not at 100 mg/kg, due to concurrent non-specific side effects. Unlike DMP696, CDP had no effect on the stress-induced increase in corticosterone at lower doses, but resulted in a significant increase at higher doses. DMP696 did not reduce locomotor activity or impair motor coordination at doses up to 30-fold higher than doses effective in the Defensive Withdrawal model. In contrast, CDP produced significant sedation and ataxia at the same doses that were effective in reducing exit latency. CONCLUSIONS: These data suggest that the CRF(1) antagonist DMP696 might retain the therapeutic benefits of classical benzodiazepines but have fewer motoric side effects.

The curious case of miRNAs in circulation: potential diagnostic biomarkers?

The pervasive occurrence of cell-free miRNAs in circulation suggests that these species play an emerging role as regulatory molecules in the secretory environment. Are these molecules released fortuitously with no clear biological intent? Or do they constitute a regulatory architecture that has evolved to modulate gene expression using the highways and byways of the circulatory system? The study of circulating miRNAs continues to increase our understanding of the regulation of genomes. The diversity of acellular miRNAs from a functional perspective is discussed, and in particular we explore their utility in a clinical setting as blood-based biomarkers for diseases.

Genome-wide maps of circulating miRNA biomarkers for ulcerative colitis.

Inflammatory Bowel Disease--comprised of Crohn's Disease and Ulcerative Colitis (UC)--is a complex, multi-factorial inflammatory disorder of the gastrointestinal tract. In this study we have explored the utility of naturally occurring circulating miRNAs as potential blood-based biomarkers for non-invasive prediction of UC incidences. Whole genome maps of circulating miRNAs in micro-vesicles, Peripheral Blood Mononuclear Cells and platelets have been constructed from a cohort of 20 UC patients and 20 normal individuals. Through Significance Analysis of Microarrays, a signature of 31 differentially expressed platelet-derived miRNAs has been identified and biomarker performance estimated through a non-probabilistic binary linear classification using Support Vector Machines. Through this approach, classifier measurements reveal a predictive score of 92.8% accuracy, 96.2% specificity and 89.5% sensitivity in distinguishing UC patients from normal individuals. Additionally, the platelet-derived biomarker signature can be validated at 88% accuracy through qPCR assays, and a majority of the miRNAs in this panel can be demonstrated to sub-stratify into 4 highly correlated intensity based clusters. Analysis of predicted targets of these biomarkers reveal an enrichment of pathways associated with cytoskeleton assembly, transport, membrane permeability and regulation of transcription factors engaged in a variety of regulatory cascades that are consistent with a cell-mediated immune response model of intestinal inflammation. Interestingly, comparison of the miRNA biomarker panel and genetic loci implicated in IBD through genome-wide association studies identifies a physical linkage between hsa-miR-941 and a UC susceptibility loci located on Chr 20. Taken together, analysis of these expression maps outlines a promising catalog of novel platelet-derived miRNA biomarkers of clinical utility and provides insight into the potential biological function of these candidates in disease pathogenesis.