Extensive sequencing of seven human genomes to characterize benchmark reference materials.

The Genome in a Bottle Consortium, hosted by the National Institute of Standards and Technology (NIST) is creating reference materials and data for human genome sequencing, as well as methods for genome comparison and benchmarking. Here, we describe a large, diverse set of sequencing data for seven human genomes; five are current or candidate NIST Reference Materials. The pilot genome, NA12878, has been released as NIST RM 8398. We also describe data from two Personal Genome Project trios, one of Ashkenazim Jewish ancestry and one of Chinese ancestry. The data come from 12 technologies: BioNano Genomics, Complete Genomics paired-end and LFR, Ion Proton exome, Oxford Nanopore, Pacific Biosciences, SOLiD, 10X Genomics GemCode WGS, and Illumina exome and WGS paired-end, mate-pair, and synthetic long reads. Cell lines, DNA, and data from these individuals are publicly available. Therefore, we expect these data to be useful for revealing novel information about the human genome and improving sequencing technologies, SNP, indel, and structural variant calling, and de novo assembly.

A hybrid approach for de novo human genome sequence assembly and phasing.

Despite tremendous progress in genome sequencing, the basic goal of producing a phased (haplotype-resolved) genome sequence with end-to-end contiguity for each chromosome at reasonable cost and effort is still unrealized. In this study, we describe an approach to performing de novo genome assembly and experimental phasing by integrating the data from Illumina short-read sequencing, 10X Genomics linked-read sequencing, and BioNano Genomics genome mapping to yield a high-quality, phased, de novo assembled human genome.

Whole-genome mutational burden analysis of three pluripotency induction methods.

There is concern that the stresses of inducing pluripotency may lead to deleterious DNA mutations in induced pluripotent stem cell (iPSC) lines, which would compromise their use for cell therapies. Here we report comparative genomic analysis of nine isogenic iPSC lines generated using three reprogramming methods: integrating retroviral vectors, non-integrating Sendai virus and synthetic mRNAs. We used whole-genome sequencing and de novo genome mapping to identify single-nucleotide variants, insertions and deletions, and structural variants. Our results show a moderate number of variants in the iPSCs that were not evident in the parental fibroblasts, which may result from reprogramming. There were only small differences in the total numbers and types of variants among different reprogramming methods. Most importantly, a thorough genomic analysis showed that the variants were generally benign. We conclude that the process of reprogramming is unlikely to introduce variants that would make the cells inappropriate for therapy.

Genome-Wide Structural Variation Detection by Genome Mapping on Nanochannel Arrays.

Comprehensive whole-genome structural variation detection is challenging with current approaches. With diploid cells as DNA source and the presence of numerous repetitive elements, short-read DNA sequencing cannot be used to detect structural variation efficiently. In this report, we show that genome mapping with long, fluorescently labeled DNA molecules imaged on nanochannel arrays can be used for whole-genome structural variation detection without sequencing. While whole-genome haplotyping is not achieved, local phasing (across >150-kb regions) is routine, as molecules from the parental chromosomes are examined separately. In one experiment, we generated genome maps from a trio from the 1000 Genomes Project, compared the maps against that derived from the reference human genome, and identified structural variations that are >5 kb in size. We find that these individuals have many more structural variants than those published, including some with the potential of disrupting gene function or regulation.

Noninvasive prenatal detection of sex chromosomal aneuploidies by sequencing circulating cell-free DNA from maternal plasma.

OBJECTIVE: Whole-genome sequencing of circulating cell free (ccf) DNA from maternal plasma has enabled noninvasive prenatal testing for common autosomal aneuploidies. The purpose of this study was to extend the detection to include common sex chromosome aneuploidies (SCAs): [47,XXX], [45,X], [47,XXY], and [47,XYY] syndromes. METHOD: Massively parallel sequencing was performed on ccf DNA isolated from the plasma of 1564 pregnant women with known fetal karyotype. A classification algorithm for SCA detection was constructed and trained on this cohort. Another study of 411 maternal samples from women with blinded-to-laboratory fetal karyotypes was then performed to determine the accuracy of the classification algorithm. RESULTS: In the training cohort, the new algorithm had a detection rate (DR) of 100% (95%CI: 82.3%, 100%), a false positive rate (FPR) of 0.1% (95%CI: 0%, 0.3%), and nonreportable rate of 6% (95%CI: 4.9%, 7.4%) for SCA determination. The blinded validation yielded similar results: DR of 96.2% (95%CI: 78.4%, 99.8%), FPR of 0.3% (95%CI: 0%, 1.8%), and nonreportable rate of 5% (95%CI: 3.2%, 7.7%) for SCA determination CONCLUSION: Noninvasive prenatal identification of the most common sex chromosome aneuploidies is possible using ccf DNA and massively parallel sequencing with a high DR and a low FPR.

High-throughput massively parallel sequencing for fetal aneuploidy detection from maternal plasma.

BACKGROUND: Circulating cell-free (ccf) fetal DNA comprises 3-20% of all the cell-free DNA present in maternal plasma. Numerous research and clinical studies have described the analysis of ccf DNA using next generation sequencing for the detection of fetal aneuploidies with high sensitivity and specificity. We sought to extend the utility of this approach by assessing semi-automated library preparation, higher sample multiplexing during sequencing, and improved bioinformatic tools to enable a higher throughput, more efficient assay while maintaining or improving clinical performance. METHODS: Whole blood (10mL) was collected from pregnant female donors and plasma separated using centrifugation. Ccf DNA was extracted using column-based methods. Libraries were prepared using an optimized semi-automated library preparation method and sequenced on an Illumina HiSeq2000 sequencer in a 12-plex format. Z-scores were calculated for affected chromosomes using a robust method after normalization and genomic segment filtering. Classification was based upon a standard normal transformed cutoff value of z = 3 for chromosome 21 and z = 3.95 for chromosomes 18 and 13. RESULTS: Two parallel assay development studies using a total of more than 1900 ccf DNA samples were performed to evaluate the technical feasibility of automating library preparation and increasing the sample multiplexing level. These processes were subsequently combined and a study of 1587 samples was completed to verify the stability of the process-optimized assay. Finally, an unblinded clinical evaluation of 1269 euploid and aneuploid samples utilizing this high-throughput assay coupled to improved bioinformatic procedures was performed. We were able to correctly detect all aneuploid cases with extremely low false positive rates of 0.09%, <0.01%, and 0.08% for trisomies 21, 18, and 13, respectively. CONCLUSIONS: These data suggest that the developed laboratory methods in concert with improved bioinformatic approaches enable higher sample throughput while maintaining high classification accuracy.

Detection of microdeletion 22q11.2 in a fetus by next-generation sequencing of maternal plasma.

BACKGROUND: Efforts have been undertaken recently to assess the fetal genome through analysis of circulating cell-free (ccf) fetal DNA obtained from maternal plasma. Sequencing analysis of such ccf DNA has been shown to enable accurate prenatal detection of fetal aneuploidies, including trisomies of chromosomes 21, 18, and 13. We sought to extend these analyses to examine subchromosomal copy number variants through the sequencing of ccf DNA. We examined a clinically relevant genomic region, chromosome 22q11.2, the location of a series of well-characterized deletion anomalies that cause 22q11.2 deletion syndrome. METHODS: We sequenced ccf DNA isolated from maternal plasma samples obtained from 2 patients with confirmed 22q11.2 deletion syndrome and from 14 women at low risk for fetal chromosomal abnormalities. The latter samples were used as controls, and the mean genomic coverage was 3.83-fold. Data were aligned to the human genome, repetitive regions were removed, the remaining data were normalized for GC content, and z scores were calculated for the affected region. RESULTS: The median fetal DNA contribution for all samples was 18%, with the affected samples containing 17%-18% fetal DNA. Using a technique similar to that used for sequencing-based fetal aneuploidy detection from maternal plasma, we detected a statistically significant loss of representation of a portion of chromosome 22q11.2 in both of the affected fetal samples. No such loss was detected in any of the control samples. CONCLUSIONS: Noninvasive prenatal diagnosis of subchromosomal fetal genomic anomalies is feasible with next-generation sequencing.

Brain energetics and tolerance to anoxia in deep hypothermia.

The remarkable time-resolution enhancement by deep lethargic hypothermia (15 degrees C rectal temperature, "cold narcosis," "anesthesia by internal cold") of metabolic events in the rat brain after oxygen deprivation has been exploited to monitor metabolic changes by in vivo (31)P-NMR. A correlation was established between the bioenergetic status of the brain and physiological descriptors of tolerance (survival and revival times) determined in parallel experiments with large series of animals. Spectral peak integrals were transformed into absolute concentrations by comparison to biochemically determined time series of data obtained in freeze-trapping experiments conducted under identical conditions. Serial spectra were used to reconstruct the time-course kinetics of intracellular brain pH and of concentration changes of inorganic phosphate, phosphocreatine, ATP, and ADP. Both the biochemical and NMR time series of data were simultaneously fitted by a set of exponential kinetic equations accounting for relationships imposed by the Lohmann and adenylate kinase reactions. Depletion profiles were then computed for a number of descriptors of brain energy status (energy charge, phosphorylation potential, total adenylate, and primary energy stores expressed as the sum of high-energy phosphate-bond equivalents). The results contribute to the understanding of the role of brain energetics in tolerance to oxygen deprivation.

Equilibrium of nucleotides in the dogfish brain.

In the past, the results of experiments on the time course of concentration changes of adenylates, phosphocreatine, and free creatine in muscle appeared compatible with an equilibrium hypothesis involving only the Lohmann and the myokinase reactions. Other reports, however, denied the applicability of the equilibrium hypothesis to the same tissue. The controversy may have been due to the high probability of experimental errors since time sampling was performed at second intervals. We presently test the hypothesis in the living brain of the small-spotted dogfish shark (Scyliorhinus canicula), an animal-model allowing for timing of sampling at hourly intervals. According to our earlier work, the dogfish shark can easily be resuscitated 8.2 h on average after being brought into the state of "suspended animation" at 0 degree C body temperature and exposed, out of water, to an atmosphere of nitrogen gas. To obtain a complete mathematical description of the time course of concentration changes of brain adenylates and phosphocreatine, we devised a kinetic model based on principles of classical multicompartmental analysis and biochemical kinetics. Model testing of the equilibrium hypothesis resulted in very good agreement between the hypothesis and our experimental data. Time-course modeling, achieved by simultaneously fitting the time series of our data by the set of four equations constituting our model resulted in an excellent agreement between data points and the computed curves. Finally, modeling of the depletion profiles of brain energy status concerning three of its descriptors (energy charge, total adenylate, and primary energy stores expressed in high-energy phosphate equivalents) allowed for a correlation to be established between energy status and the "revival time," a valuable physiological descriptor of tolerance.

Mechanisms of immediate temperature compensation: experiments with brain synaptosomes from rat and ground squirrel.

Glucose conversion by brain synaptosomes can be regarded as a special case of intrinsic kinetic properties of the enzyme substrate system. Temperature modulation of apparent K(m) for this process can be described with our kinetic model. Using experimental data and the kinetic model, the minimal K(m) value for glucose conversion in ground squirrel synaptosomes was found at the lower temperature (6.5 degrees C), much lower than that for the rat (16.6 degrees C). The inversion temperatures (T(min)) closely coincided with the lowest body temperatures from which the unassisted recovery from hypothermia was demonstrated in both species. This study indicated that thermal modulation of enzyme affinities may have an adaptive role in endotherms that is linked to their tolerance to hypothermia.

Kinetic properties of the enzyme-substrate system: a basis for immediate temperature compensation.

One-minimum U-shaped temperature profiles of the dissociation constant (K(m)) have been observed experimentally with a variety of enzyme-substrate (E-S) systems. The increase of E-S affinity with falling temperature ("positive thermal modulation of affinity"), which opposes the cold-induced reduction in catalytic velocity, has been often interpreted as significant for both immediate and evolutionary temperature compensations and of major importance in setting thermal limits in ectothermic organisms. This role was denied to enzymes from endotherms, on the ground that their minimal K(m) values were situated well below their normal body temperature. Evidence is presented in this report that affinity changes described by U-shaped profiles can simply be the consequence of intrinsic kinetic properties of the E-S system. Theoretical modeling is achieved by combining the classical expression for the Michaelis constant with Transition State Theory expressions for the three rate constants involved. It provides for the U-shape of the K(m) vs. T profile and allows for the derivation of an equation for identifying its inversion point. Modeling of V(max) and V(min) (reaction velocity under conditions of substrate saturation and of dilution, K(m)>>[S], respectively) is also included. An expression was formulated for predicting the "critical temperature," T(C), corresponding to the low-temperature break in Arrhenius lines. Using existing K(m) data from literature, concerning a variety of E-S systems, our modeling proved to be highly satisfactory. Our own experiments show that glucose uptake by rat brain synaptosomes can be regarded as a special case of basically the same kinetic scheme, and that the U-shaped temperature modulation of apparent K(m) for glucose conversion is also in full agreement with our kinetic modeling. These experiments indicate that positive thermal modulation, although based on intrinsic kinetic properties of the underlying E-S system, may have an adaptive role in endotherms as well, linked, however, to their tolerance to hypothermia.