New realm of precision multiplexing enabled by massively-parallel single molecule UltraPCR.

PCR has been a reliable and inexpensive method for nucleic acid detection in the past several decades. In particular, multiplex PCR is a powerful tool to analyze many biomarkers in the same reaction, thus maximizing detection sensitivity and reducing sample usage. However, balancing the amplification kinetics between amplicons and distinguishing them can be challenging, diminishing the broad adoption of high order multiplex PCR panels. Here, we present a new paradigm in PCR amplification and multiplexed detection using UltraPCR. UltraPCR utilizes a simple centrifugation workflow to split a PCR reaction into ∼34 million partitions, forming an optically clear pellet of spatially separated reaction compartments in a PCR tube. After in situ thermocycling, light sheet scanning is used to produce a 3D reconstruction of the fluorescent positive compartments within the pellet. At typical sample DNA concentrations, the magnitude of partitions offered by UltraPCR dictate that the vast majority of target molecules occupy a compartment uniquely. This single molecule realm allows for isolated amplification events, thereby eliminating competition between different targets and generating unambiguous optical signals for detection. Using a 4-color optical setup, we demonstrate that we can incorporate 10 different fluorescent dyes in the same UltraPCR reaction. We further push multiplexing to an unprecedented level by combinatorial labeling with fluorescent dyes - referred to as "comboplex" technology. Using the same 4-color optical setup, we developed a 22-target comboplex panel that can detect all targets simultaneously at high precision. Collectively, UltraPCR has the potential to push PCR applications beyond what is currently available, enabling a new class of precision genomics assays.

Next-Generation Digital Polymerase Chain Reaction: High-Dynamic-Range Single-Molecule DNA Counting via Ultrapartitioning.

Digital PCR (dPCR) was first conceived for single-molecule quantitation. However, current dPCR systems often require DNA templates to share partitions due to limited partitioning capacities. Here, we introduce UltraPCR, a next-generation dPCR system where DNA counting is performed in a single-molecule regimen through a 6-log dynamic range using a swift and parallelized workflow. Each UltraPCR reaction is divided into >30 million partitions without microfluidics to achieve single template occupancy. Combined with a unique emulsion chemistry, partitions are optically clear, enabling the use of a three-dimensional imaging technique to rapidly detect DNA-positive partitions. Single-molecule occupancy also allows for more straightforward multiplex assay development due to the absence of partition-specific competition. As a proof of concept, we developed a 222-plex UltraPCR assay and demonstrated its potential use as a rapid, low-cost screening assay for noninvasive prenatal testing for as low as 4% trisomy fraction samples with high precision, accuracy, and reproducibility.

Generation of Functional Human 3D Cortico-Motor Assembloids.

Neurons in the cerebral cortex connect through descending pathways to hindbrain and spinal cord to activate muscle and generate movement. Although components of this pathway have been previously generated and studied in vitro, the assembly of this multi-synaptic circuit has not yet been achieved with human cells. Here, we derive organoids resembling the cerebral cortex or the hindbrain/spinal cord and assemble them with human skeletal muscle spheroids to generate 3D cortico-motor assembloids. Using rabies tracing, calcium imaging, and patch-clamp recordings, we show that corticofugal neurons project and connect with spinal spheroids, while spinal-derived motor neurons connect with muscle. Glutamate uncaging or optogenetic stimulation of cortical spheroids triggers robust contraction of 3D muscle, and assembloids are morphologically and functionally intact for up to 10 weeks post-fusion. Together, this system highlights the remarkable self-assembly capacity of 3D cultures to form functional circuits that could be used to understand development and disease.

Reliability of human cortical organoid generation.

The differentiation of pluripotent stem cells in three-dimensional cultures can recapitulate key aspects of brain development, but protocols are prone to variable results. Here we differentiated multiple human pluripotent stem cell lines for over 100 d using our previously developed approach to generate brain-region-specific organoids called cortical spheroids and, using several assays, found that spheroid generation was highly reliable and consistent. We anticipate the use of this approach for large-scale differentiation experiments and disease modeling.

An interpretable framework for clustering single-cell RNA-Seq datasets.

BACKGROUND: With the recent proliferation of single-cell RNA-Seq experiments, several methods have been developed for unsupervised analysis of the resulting datasets. These methods often rely on unintuitive hyperparameters and do not explicitly address the subjectivity associated with clustering. RESULTS: In this work, we present DendroSplit, an interpretable framework for analyzing single-cell RNA-Seq datasets that addresses both the clustering interpretability and clustering subjectivity issues. DendroSplit offers a novel perspective on the single-cell RNA-Seq clustering problem motivated by the definition of "cell type", allowing us to cluster using feature selection to uncover multiple levels of biologically meaningful populations in the data. We analyze several landmark single-cell datasets, demonstrating both the method's efficacy and computational efficiency. CONCLUSION: DendroSplit offers a clustering framework that is comparable to existing methods in terms of accuracy and speed but is novel in its emphasis on interpretabilty. We provide the full DendroSplit software package at https://github.com/jessemzhang/dendrosplit .

Assembly of functionally integrated human forebrain spheroids.

The development of the nervous system involves a coordinated succession of events including the migration of GABAergic (γ-aminobutyric-acid-releasing) neurons from ventral to dorsal forebrain and their integration into cortical circuits. However, these interregional interactions have not yet been modelled with human cells. Here we generate three-dimensional spheroids from human pluripotent stem cells that resemble either the dorsal or ventral forebrain and contain cortical glutamatergic or GABAergic neurons. These subdomain-specific forebrain spheroids can be assembled in vitro to recapitulate the saltatory migration of interneurons observed in the fetal forebrain. Using this system, we find that in Timothy syndrome-a neurodevelopmental disorder that is caused by mutations in the CaV1.2 calcium channel-interneurons display abnormal migratory saltations. We also show that after migration, interneurons functionally integrate with glutamatergic neurons to form a microphysiological system. We anticipate that this approach will be useful for studying neural development and disease, and for deriving spheroids that resemble other brain regions to assemble circuits in vitro.

B cell repertoires in HLA-sensitized kidney transplant candidates undergoing desensitization therapy.

BACKGROUND: Kidney transplantation is the most effective treatment for end-stage renal disease. Sensitization refers to pre-existing antibodies against human leukocyte antigen (HLA) protein and remains a major barrier to successful transplantation. Despite implementation of desensitization strategies, many candidates fail to respond. Our objective was to determine whether measuring B cell repertoires could differentiate candidates that respond to desensitization therapy. METHODS: We developed an assay based on high-throughput DNA sequencing of the variable domain of the heavy chain of immunoglobulin genes to measure changes in B cell repertoires in 19 highly HLA-sensitized kidney transplant candidates undergoing desensitization and 7 controls with low to moderate HLA sensitization levels. Responders to desensitization had a decrease of 5% points or greater in cumulated calculated panel reactive antibody (cPRA) levels, and non-responders had no decrease in cPRA. RESULTS: Dominant B cell clones were not observed in highly sensitized candidates, suggesting that the B cells responsible for sensitization are either not present in peripheral blood or present at comparable levels to other circulating B cells. Candidates that responded to desensitization therapy had pre-treatment repertoires composed of a larger fraction of class-switched (IgG and IgA) isotypes compared to non-responding candidates. After B cell depleting therapy, the proportion of switched isotypes increased and the mutation frequencies of the remaining non-switched isotypes (IgM and IgD) increased in both responders and non-responders, perhaps representing a shift in the repertoire towards memory B cells or plasmablasts. Conversely, after transplantation, non-switched isotypes with fewer mutations increased, suggesting a shift in the repertoire towards naïve B cells. CONCLUSIONS: Relative abundance of different B cell isotypes is strongly perturbed by desensitization therapy and transplantation, potentially reflecting changes in the relative abundance of memory and naïve B cell compartments. Candidates that responded to therapy experienced similar changes to those that did not respond. Further studies are required to understand differences between these two groups of highly sensitized kidney transplant candidates.

Expression profiling. Combinatorial labeling of single cells for gene expression cytometry.

We present a technically simple approach for gene expression cytometry combining next-generation sequencing with stochastic barcoding of single cells. A combinatorial library of beads bearing cell- and molecular-barcoding capture probes is used to uniquely label transcripts and reconstruct the digital gene expression profile of thousands of individual cells in a single experiment without the need for robotics or automation. We applied the technology to dissect the human hematopoietic system and to characterize heterogeneous response to in vitro stimulation. High sensitivity is demonstrated by detection of low-abundance transcripts and rare cells. Under current implementation, the technique can analyze a few thousand cells simultaneously and can readily scale to 10,000s or 100,000s of cells.

Noninvasive in vivo monitoring of tissue-specific global gene expression in humans.

Circulating cell-free RNA in the blood provides a potential window into the health, phenotype, and developmental programs of a variety of human organs. We used high-throughput methods of RNA analysis such as microarrays and next-generation sequencing to characterize the global landscape circulating RNA in a cohort of human subjects. By focusing on genes whose expression is highly specific to certain tissues, we were able to identify the relative contributions of these tissues to circulating RNA and to monitor changes in tissue development and health. As one application of this approach, we performed a longitudinal study on pregnant women and analyzed their combined cell-free RNA transcriptomes across all three trimesters of pregnancy and after delivery. In addition to the analysis of mRNA, we observed and characterized noncoding species such as long noncoding RNA and circular RNA transcripts whose presence had not been previously observed in human plasma. We demonstrate that it is possible to track specific longitudinal phenotypic changes in both the mother and the fetus and that it is possible to directly measure transcripts from a variety of fetal tissues in the maternal blood sample. We also studied the role of neuron-specific transcripts in the blood of healthy adults and those suffering from the neurodegenerative disorder Alzheimer's disease and showed that disease specific neural transcripts are present at increased levels in the blood of affected individuals. Characterization of the cell-free transcriptome in its entirety may thus provide broad insights into human health and development without the need for invasive tissue sampling.

Digital encoding of cellular mRNAs enabling precise and absolute gene expression measurement by single-molecule counting.

We present a new approach for the sensitive detection and accurate quantitation of messenger ribonucleic acid (mRNA) gene transcripts in single cells. First, the entire population of mRNAs is encoded with molecular barcodes during reverse transcription. After amplification of the gene targets of interest, molecular barcodes are counted by sequencing or scored on a simple hybridization detector to reveal the number of molecules in the starting sample. Since absolute quantities are measured, calibration to standards is unnecessary, and many of the relative quantitation challenges such as polymerase chain reaction (PCR) bias are avoided. We apply the method to gene expression analysis of minute sample quantities and demonstrate precise measurements with sensitivity down to sub single-cell levels. The method is an easy, single-tube, end point assay utilizing standard thermal cyclers and PCR reagents. Accurate and precise measurements are obtained without any need for cycle-to-cycle intensity-based real-time monitoring or physical partitioning into multiple reactions (e.g., digital PCR). Further, since all mRNA molecules are encoded with molecular barcodes, amplification can be used to generate more material for multiple measurements and technical replicates can be carried out on limited samples. The method is particularly useful for small sample quantities, such as single-cell experiments. Digital encoding of cellular content preserves true abundance levels and overcomes distortions introduced by amplification.

Identification of a colonial chordate histocompatibility gene.

Histocompatibility is the basis by which multicellular organisms of the same species distinguish self from nonself. Relatively little is known about the mechanisms underlying histocompatibility reactions in lower organisms. Botryllus schlosseri is a colonial urochordate, a sister group of vertebrates, that exhibits a genetically determined natural transplantation reaction, whereby self-recognition between colonies leads to formation of parabionts with a common vasculature, whereas rejection occurs between incompatible colonies. Using genetically defined lines, whole-transcriptome sequencing, and genomics, we identified a single gene that encodes self-nonself and determines "graft" outcomes in this organism. This gene is significantly up-regulated in colonies poised to undergo fusion and/or rejection, is highly expressed in the vasculature, and is functionally linked to histocompatibility outcomes. These findings establish a platform for advancing the science of allorecognition.

The genome sequence of the colonial chordate, Botryllus schlosseri.

Botryllus schlosseri is a colonial urochordate that follows the chordate plan of development following sexual reproduction, but invokes a stem cell-mediated budding program during subsequent rounds of asexual reproduction. As urochordates are considered to be the closest living invertebrate relatives of vertebrates, they are ideal subjects for whole genome sequence analyses. Using a novel method for high-throughput sequencing of eukaryotic genomes, we sequenced and assembled 580 Mbp of the B. schlosseri genome. The genome assembly is comprised of nearly 14,000 intron-containing predicted genes, and 13,500 intron-less predicted genes, 40% of which could be confidently parceled into 13 (of 16 haploid) chromosomes. A comparison of homologous genes between B. schlosseri and other diverse taxonomic groups revealed genomic events underlying the evolution of vertebrates and lymphoid-mediated immunity. The B. schlosseri genome is a community resource for studying alternative modes of reproduction, natural transplantation reactions, and stem cell-mediated regeneration. DOI:http://dx.doi.org/10.7554/eLife.00569.001.

Genome-wide single-cell analysis of recombination activity and de novo mutation rates in human sperm.

Meiotic recombination and de novo mutation are the two main contributions toward gamete genome diversity, and many questions remain about how an individual human's genome is edited by these two processes. Here, we describe a high-throughput method for single-cell whole-genome analysis that was used to measure the genomic diversity in one individual's gamete genomes. A microfluidic system was used for highly parallel sample processing and to minimize nonspecific amplification. High-density genotyping results from 91 single cells were used to create a personal recombination map, which was consistent with population-wide data at low resolution but revealed significant differences from pedigree data at higher resolution. We used the data to test for meiotic drive and found evidence for gene conversion. High-throughput sequencing on 31 single cells was used to measure the frequency of large-scale genome instability, and deeper sequencing of eight single cells revealed de novo mutation rates with distinct characteristics.

Non-invasive prenatal measurement of the fetal genome.

The vast majority of prenatal genetic testing requires invasive sampling. However, this poses a risk to the fetus, so one must make a decision that weighs the desire for genetic information against the risk of an adverse outcome due to hazards of the testing process. These issues are not required to be coupled, and it would be desirable to discover genetic information about the fetus without incurring a health risk. Here we demonstrate that it is possible to non-invasively sequence the entire prenatal genome. Our results show that molecular counting of parental haplotypes in maternal plasma by shotgun sequencing of maternal plasma DNA allows the inherited fetal genome to be deciphered non-invasively. We also applied the counting principle directly to each allele in the fetal exome by performing exome capture on maternal plasma DNA before shotgun sequencing. This approach enables non-invasive exome screening of clinically relevant and deleterious alleles that were paternally inherited or had arisen as de novo germline mutations, and complements the haplotype counting approach to provide a comprehensive view of the fetal genome. Non-invasive determination of the fetal genome may ultimately facilitate the diagnosis of all inherited and de novo genetic disease.

The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line.

Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most of the assembled scaffolds with 21 chromosomes isolated by microfluidics to identify chromosomal locations of genes. Furthermore, we investigate genes involved in glycosylation, which affect therapeutic protein quality, and viral susceptibility genes, which are relevant to cell engineering and regulatory concerns. Homologs of most human glycosylation-associated genes are present in the CHO-K1 genome, although 141 of these homologs are not expressed under exponential growth conditions. Many important viral entry genes are also present in the genome but not expressed, which may explain the unusual viral resistance property of CHO cell lines. We discuss how the availability of this genome sequence may facilitate genome-scale science for the optimization of biopharmaceutical protein production.

Whole-genome molecular haplotyping of single cells.

Conventional experimental methods of studying the human genome are limited by the inability to independently study the combination of alleles, or haplotype, on each of the homologous copies of the chromosomes. We developed a microfluidic device capable of separating and amplifying homologous copies of each chromosome from a single human metaphase cell. Single-nucleotide polymorphism (SNP) array analysis of amplified DNA enabled us to achieve completely deterministic, whole-genome, personal haplotypes of four individuals, including a HapMap trio with European ancestry (CEU) and an unrelated European individual. The phases of alleles were determined at ∼99.8% accuracy for up to ∼96% of all assayed SNPs. We demonstrate several practical applications, including direct observation of recombination events in a family trio, deterministic phasing of deletions in individuals and direct measurement of the human leukocyte antigen haplotypes of an individual. Our approach has potential applications in personal genomics, single-cell genomics and statistical genetics.

Analysis of the size distributions of fetal and maternal cell-free DNA by paired-end sequencing.

BACKGROUND: Noninvasive prenatal diagnosis with cell-free DNA in maternal plasma is challenging because only a small portion of the DNA sample is derived from the fetus. A few previous studies provided size-range estimates of maternal and fetal DNA, but direct measurement of the size distributions is difficult because of the small quantity of cell-free DNA. METHODS: We used high-throughput paired-end sequencing to directly measure the size distributions of maternal and fetal DNA in cell-free maternal plasma collected from 3 typical diploid and 4 aneuploid male pregnancies. As a control, restriction fragments of lambda DNA were also sequenced. RESULTS: Cell-free DNA had a dominant peak at approximately 162 bp and a minor peak at approximately 340 bp. Chromosome Y sequences were rarely longer than 250 bp but were present in sizes of <150 bp at a larger proportion compared with the rest of the sequences. Selective analysis of the shortest fragments generally increased the fetal DNA fraction but did not necessarily increase the sensitivity of aneuploidy detection, owing to the reduction in the number of DNA molecules being counted. Restriction fragments of lambda DNA with sizes between 60 bp and 120 bp were preferentially sequenced, indicating that the shotgun sequencing work flow introduced a bias toward shorter fragments. CONCLUSIONS: Our results confirm that fetal DNA is shorter than maternal DNA. The enrichment of fetal DNA by size selection, however, may not provide a dramatic increase in sensitivity for assays that rely on length measurement in situ because of a trade-off between the fetal DNA fraction and the number of molecules being counted.

Sensitivity of noninvasive prenatal detection of fetal aneuploidy from maternal plasma using shotgun sequencing is limited only by counting statistics.

We recently demonstrated noninvasive detection of fetal aneuploidy by shotgun sequencing cell-free DNA in maternal plasma using next-generation high throughput sequencer. However, GC bias introduced by the sequencer placed a practical limit on the sensitivity of aneuploidy detection. In this study, we describe a method to computationally remove GC bias in short read sequencing data by applying weight to each sequenced read based on local genomic GC content. We show that sensitivity is limited only by counting statistics and that sensitivity can be increased to arbitrary precision in sample containing arbitrarily small fraction of fetal DNA simply by sequencing more DNA molecules. High throughput shotgun sequencing of maternal plasma DNA should therefore enable noninvasive diagnosis of any type of fetal aneuploidy.

Microfluidic digital PCR enables rapid prenatal diagnosis of fetal aneuploidy.

OBJECTIVE: The purpose of this study was to demonstrate that digital polymerase chain reaction (PCR) enables rapid, allele independent molecular detection of fetal aneuploidy. STUDY DESIGN: Twenty-four amniocentesis and 16 chorionic villus samples were used for microfluidic digital PCR analysis. Three thousand and sixty PCR reactions were performed for each of the target chromosomes (X, Y, 13, 18, and 21), and the number of single molecule amplifications was compared to a reference. The difference between target and reference chromosome counts was used to determine the ploidy of each of the target chromosomes. RESULTS: Digital PCR accurately identified all cases of fetal trisomy (3 cases of trisomy 21, 3 cases of trisomy 18, and 2 cases of triosmy 13) in the 40 specimens analyzed. The remaining specimens were determined to have normal ploidy for the chromosomes tested. CONCLUSION: Microfluidic digital PCR allows detection of fetal chromosomal aneuploidy utilizing uncultured amniocytes and chorionic villus tissue in less than 6 hours.

Digital PCR provides sensitive and absolute calibration for high throughput sequencing.

BACKGROUND: Next-generation DNA sequencing on the 454, Solexa, and SOLiD platforms requires absolute calibration of the number of molecules to be sequenced. This requirement has two unfavorable consequences. First, large amounts of sample-typically micrograms-are needed for library preparation, thereby limiting the scope of samples which can be sequenced. For many applications, including metagenomics and the sequencing of ancient, forensic, and clinical samples, the quantity of input DNA can be critically limiting. Second, each library requires a titration sequencing run, thereby increasing the cost and lowering the throughput of sequencing. RESULTS: We demonstrate the use of digital PCR to accurately quantify 454 and Solexa sequencing libraries, enabling the preparation of sequencing libraries from nanogram quantities of input material while eliminating costly and time-consuming titration runs of the sequencer. We successfully sequenced low-nanogram scale bacterial and mammalian DNA samples on the 454 FLX and Solexa DNA sequencing platforms. This study is the first to definitively demonstrate the successful sequencing of picogram quantities of input DNA on the 454 platform, reducing the sample requirement more than 1000-fold without pre-amplification and the associated bias and reduction in library depth. CONCLUSION: The digital PCR assay allows absolute quantification of sequencing libraries, eliminates uncertainties associated with the construction and application of standard curves to PCR-based quantification, and with a coefficient of variation close to 10%, is sufficiently precise to enable direct sequencing without titration runs.