Large-scale population analyses coupled with advances in technology have demonstrated that the human genome is more diverse than originally thought. To date, this diversity has largely been uncovered using short-read whole-genome sequencing. However, these short-read approaches fail to give a complete picture of a genome. They struggle to identify structural events, cannot access repetitive regions, and fail to resolve the human genome into haplotypes. Here, we describe an approach that retains long range information while maintaining the advantages of short reads. Starting from ∼1 ng of high molecular weight DNA, we produce barcoded short-read libraries. Novel informatic approaches allow for the barcoded short reads to be associated with their original long molecules producing a novel data type known as "Linked-Reads". This approach allows for simultaneous detection of small and large variants from a single library. In this manuscript, we show the advantages of Linked-Reads over standard short-read approaches for reference-based analysis. Linked-Reads allow mapping to 38 Mb of sequence not accessible to short reads, adding sequence in 423 difficult-to-sequence genes including disease-relevant genes STRC, SMN1, and SMN2 Both Linked-Read whole-genome and whole-exome sequencing identify complex structural variations, including balanced events and single exon deletions and duplications. Further, Linked-Reads extend the region of high-confidence calls by 68.9 Mb. The data presented here show that Linked-Reads provide a scalable approach for comprehensive genome analysis that is not possible using short reads alone.

ABSTRACT Characterizing the transcriptome of individual cells is fundamental to understanding complex biological systems. We describe a droplet-based system that enables 3′ mRNA counting of up to tens of thousands of single cells per sample. Cell encapsulation in droplets takes place in ∼6 minutes, with ∼50% cell capture efficiency, up to 8 samples at a time. The speed and efficiency allow the processing of precious samples while minimizing stress to cells. To demonstrate the system′s technical performance and its applications, we collected transcriptome data from ∼¼ million single cells across 29 samples. First, we validate the sensitivity of the system and its ability to detect rare populations using cell lines and synthetic RNAs. Then, we profile 68k peripheral blood mononuclear cells (PBMCs) to demonstrate the system′s ability to characterize large immune populations. Finally, we use sequence variation in the transcriptome data to determine host and donor chimerism at single cell resolution in bone marrow mononuclear cells (BMMCs) of transplant patients. This analysis enables characterization of the complex interplay between donor and host cells and monitoring of treatment response. This high-throughput system is robust and enables characterization of diverse biological systems with single cell mRNA analysis.

Haplotyping of human chromosomes is a prerequisite for cataloguing the full repertoire of genetic variation. We present a microfluidics-based, linked-read sequencing technology that can phase and haplotype germline and cancer genomes using nanograms of input DNA. This high-throughput platform prepares barcoded libraries for short-read sequencing and computationally reconstructs long-range haplotype and structural variant information. We generate haplotype blocks in a nuclear trio that are concordant with expected inheritance patterns and phase a set of structural variants. We also resolve the structure of the EML4-ALK gene fusion in the NCI-H2228 cancer cell line using phased exome sequencing. Finally, we assign genetic aberrations to specific megabase-scale haplotypes generated from whole-genome sequencing of a primary colorectal adenocarcinoma. This approach resolves haplotype information using up to 100 times less genomic DNA than some methods and enables the accurate detection of structural variants.

Summary 1/1 "compositions and methods for sample processing" This invention provides compositions and methods for processing samples, particularly for sequencing applications. Included within this disclosure are compositions granules, such as various libraries of beads bound to a large number of oligonucleotides containing barcodes. Often the beads provided herein are degradable. for example, they may contain dissulfuetos bonds which are susceptible to reducing agents. the methods provided herein include methods of preparing granules with a bar code libraries and the methods of combining beads with a sample, such as using a microfluidic device.

We demonstrate a new approach to manipulate the selective emission in mixed electrogenerated chemiluminescence (ECL) systems, where subtle changes in co-reactant properties are exploited to control the relative electron-transfer processes of excitation and quenching. Two closely related tertiary-amine co-reactants, tri-n-propylamine and N, N-diisopropylethylamine, generate remarkably different emission profiles: one provides distinct green and red ECL from [Ir(ppy)(3)] (ppy=2-phenylpyridinato-C2, N) and a [Ru(bpy)(3)](2+) (bpy=2,2'-bipyridine) derivative at different applied potentials, whereas the other generates both emissions simultaneously across a wide potential range. These phenomena can be rationalized through the relative exergonicities of electron-transfer quenching of the excited states, in conjunction with the change in concentration of the quenchers over the applied potential range.

Droplet digital PCR shows greater precision and reproducibility but no consistent gain in sensitivity when compared to real-time PCR. Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased 37–86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer.

Chemiluminescence was observed from reactions with a Mn(IV) colloid generated by reduction of KMnO4 with Na2S2O3. Due to the limited stability of the colloid under acidic conditions and the poor reproducibility of the chemiluminescence signal when the reagent was prepared by published methods, an in situ (on-line) method of preparation utilising flow injection analysis methodology was developed. This approach provided superior emission intensities to the conventional Mn(IV) reagent, although less enhancement was obtained from the addition of formaldehyde. The selectivity of the reagent was ‘tuned’ towards specific classes of analyte through convenient modification of reaction conditions. Contrary to some previous reports, we attribute this chemiluminescence to the formation of an electronically excited Mn(II) species, based on direct comparison of the spectral distribution with that of other chemiluminescence reactions.

Two years ago, we described the first droplet digital PCR (ddPCR) system aimed at empowering all researchers with a tool that removes the substantial uncertainties associated with using the analogue standard, quantitative real-time PCR (qPCR). This system enabled TaqMan hydrolysis probe-based assays for the absolute quantification of nucleic acids. Due to significant advancements in droplet chemistry and buoyed by the multiple benefits associated with dye-based target detection, we have created a "second generation" ddPCR system compatible with both TaqMan-probe and DNA-binding dye detection chemistries. Herein, we describe the operating characteristics of DNA-binding dye based ddPCR and offer a side-by-side comparison to TaqMan probe detection. By partitioning each sample prior to thermal cycling, we demonstrate that it is now possible to use a DNA-binding dye for the quantification of multiple target species from a single reaction. The increased resolution associated with partitioning also made it possible to visualize and account for signals arising from nonspecific amplification products. We expect that the ability to combine the precision of ddPCR with both DNA-binding dye and TaqMan probe detection chemistries will further enable the research community to answer complex and diverse genetic questions.

The emission of light from the permanganate-oxalate reaction enables monitoring of intermediates not accessible through traditional spectrophotometric interrogation. Despite the inherent complexity of the underlying chemical reactions and equilibria, the emission intensity-time profile was characterized by a simple model combining previously independent minimalistic descriptions of chemiluminescence and autocatalysis. The generation of the electronically excited [Mn(II)]* emitter and the acceleration of the reaction even in the presence of high initial concentrations of Mn(II) (under conditions that preclude accumulation of colloidal Mn(IV)) provide new evidence for the reduction of manganese species by a reactive radical intermediate as a supplementary positive feedback loop to the formation of Mn(II).

Droplet digital polymerase chain reaction (ddPCR) is a new technology that was recently commercialized to enable the precise quantification of target nucleic acids in a sample. ddPCR measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions. This novel ddPCR format offers a simple workflow capable of generating highly stable partitioning of DNA molecules. In this study, we assessed key performance parameters of the ddPCR system. A linear ddPCR response to DNA concentration was obtained from 0.16% through to 99.6% saturation in a 20,000 droplet assay corresponding to more than 4 orders of magnitude of target DNA copy number per ddPCR Analysis of simplex and duplex assays targeting two distinct loci in the Lambda DNA genome using the ddPCR platform agreed, within their expanded uncertainties, with values obtained using a lower density microfluidic chamber based digital PCR (cdPCR). A relative expanded uncertainty under 5% was achieved for copy number concentration using ddPCR This level of uncertainty is much lower than values typically observed for quantification of specific DNA target sequences using currently commercially available real-time and digital cdPCR technologies.

A chemically initiated adaptation of the classic [Ru(bipy)(3)](2+)/oxalate electrochemiluminescence coreactant system has revealed the elusive radical intermediates of the light-producing pathway. Oxalyl (HC2O4 center dot) and hydroxyformyl (HCO2 center dot) radicals have been captured on a quartz surface and characterised using EPR spectroscopy.

Chemistry – A European JournalVolume 17, Issue 29 p. 8018-8022 Communication Any Old Radical Won't Do: An EPR Study of the Selective Excitation and Quenching Mechanisms of [Ru(bipy)3]2+ Chemiluminescence and Electrochemiluminescence Christopher M. Hindson, Christopher M. Hindson Deakin University, School of Life and Environmental Sciences, Pigdons Road, Waurn Ponds, Victoria 3217 (Australia), Fax: (+61) 3-5227-1040Search for more papers by this authorProf. Graeme R. Hanson, Corresponding Author Prof. Graeme R. Hanson [email protected] The University of Queensland, Centre for Advanced Imaging, St. Lucia, Queensland 4072 (Australia), Fax: (+61) 7-3365-3833 Graeme R. Hanson, The University of Queensland, Centre for Advanced Imaging, St. Lucia, Queensland 4072 (Australia), Fax: (+61) 7-3365-3833 Neil W. Barnett, Deakin University, School of Life and Environmental Sciences, Pigdons Road, Waurn Ponds, Victoria 3217 (Australia), Fax: (+61) 3-5227-1040Search for more papers by this authorDr. Paul S. Francis, Dr. Paul S. Francis Deakin University, School of Life and Environmental Sciences, Pigdons Road, Waurn Ponds, Victoria 3217 (Australia), Fax: (+61) 3-5227-1040Search for more papers by this authorDr. Jacqui L. Adcock, Dr. Jacqui L. Adcock Deakin University, School of Life and Environmental Sciences, Pigdons Road, Waurn Ponds, Victoria 3217 (Australia), Fax: (+61) 3-5227-1040Search for more papers by this authorProf. Neil W. Barnett, Corresponding Author Prof. Neil W. Barnett [email protected] Deakin University, School of Life and Environmental Sciences, Pigdons Road, Waurn Ponds, Victoria 3217 (Australia), Fax: (+61) 3-5227-1040 Graeme R. Hanson, The University of Queensland, Centre for Advanced Imaging, St. Lucia, Queensland 4072 (Australia), Fax: (+61) 7-3365-3833 Neil W. Barnett, Deakin University, School of Life and Environmental Sciences, Pigdons Road, Waurn Ponds, Victoria 3217 (Australia), Fax: (+61) 3-5227-1040Search for more papers by this author Christopher M. Hindson, Christopher M. Hindson Deakin University, School of Life and Environmental Sciences, Pigdons Road, Waurn Ponds, Victoria 3217 (Australia), Fax: (+61) 3-5227-1040Search for more papers by this authorProf. Graeme R. Hanson, Corresponding Author Prof. Graeme R. Hanson [email protected] The University of Queensland, Centre for Advanced Imaging, St. Lucia, Queensland 4072 (Australia), Fax: (+61) 7-3365-3833 Graeme R. Hanson, The University of Queensland, Centre for Advanced Imaging, St. Lucia, Queensland 4072 (Australia), Fax: (+61) 7-3365-3833 Neil W. Barnett, Deakin University, School of Life and Environmental Sciences, Pigdons Road, Waurn Ponds, Victoria 3217 (Australia), Fax: (+61) 3-5227-1040Search for more papers by this authorDr. Paul S. Francis, Dr. Paul S. Francis Deakin University, School of Life and Environmental Sciences, Pigdons Road, Waurn Ponds, Victoria 3217 (Australia), Fax: (+61) 3-5227-1040Search for more papers by this authorDr. Jacqui L. Adcock, Dr. Jacqui L. Adcock Deakin University, School of Life and Environmental Sciences, Pigdons Road, Waurn Ponds, Victoria 3217 (Australia), Fax: (+61) 3-5227-1040Search for more papers by this authorProf. Neil W. Barnett, Corresponding Author Prof. Neil W. Barnett [email protected] Deakin University, School of Life and Environmental Sciences, Pigdons Road, Waurn Ponds, Victoria 3217 (Australia), Fax: (+61) 3-5227-1040 Graeme R. Hanson, The University of Queensland, Centre for Advanced Imaging, St. Lucia, Queensland 4072 (Australia), Fax: (+61) 7-3365-3833 Neil W. Barnett, Deakin University, School of Life and Environmental Sciences, Pigdons Road, Waurn Ponds, Victoria 3217 (Australia), Fax: (+61) 3-5227-1040Search for more papers by this author First published: 07 June 2011 https://doi.org/10.1002/chem.201100877Citations: 23Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Graphical Abstract [Ru(bipy)3]2+ chemiluminescence: The mechanisms governing the selectivity of the luminescence of the [Ru(bipy)3]2+ complex (bipy=2,2′-bipyridyl) were investigated using continuous flow EPR spectroscopy. The radical intermediates of substrates thought to evoke or quench the emission from this reagent were characterized in the light-producing pathway. Supporting Information Detailed facts of importance to specialist readers are published as "Supporting Information". Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Filename Description chem_201100877_sm_miscellaneous_information.pdf81 KB miscellaneous_information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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