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 tens of thousands of single cells per sample. Cell encapsulation, of up to 8 samples at a time, takes place in ∼6 min, with ∼50% cell capture efficiency. To demonstrate the system's technical performance, we collected transcriptome data from ∼250k single cells across 29 samples. We validated the sensitivity of the system and its ability to detect rare populations using cell lines and synthetic RNAs. We profiled 68k peripheral blood mononuclear cells to demonstrate the system's ability to characterize large immune populations. Finally, we used sequence variation in the transcriptome data to determine host and donor chimerism at single-cell resolution from bone marrow mononuclear cells isolated from transplant patients.
40Characterizing the transcriptome of individual cells is fundamental to understanding complex 41 biological systems. We describe a droplet-based system that enables 3' mRNA counting of up 56peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/065912 doi: bioRxiv preprint first posted online 84 RESULTS 86Droplet-based platform enables barcoding of tens of thousands of cells 88The scRNA-seq microfluidics platform builds on the GemCode ® technology, which has 89 been used for genome haplotyping, structural variant analysis and de novo assembly of a 90human genome [10][11][12] . The core of the technology is a Gel bead in Emulsion (GEM). GEM 91 peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/065912 doi: bioRxiv preprint first posted online 5 generation takes place in an 8-channel microfluidic chip that encapsulates single gel beads at ~80% fill rate (Fig. 1a-c). Each gel bead is functionalized with barcoded oligonucleotides that 93 consist of: i) sequencing adapters and primers, ii) a 14bp barcode drawn from ~750,000 94 designed sequences to index GEMs, iii) a 10bp randomer to index molecules (unique molecular 95 identifier, UMI), and iv) an anchored 30bp oligo-dT to prime poly-adenylated RNA transcripts 96 (Fig. 1d). Within each microfluidic channel, ~100,000 GEMs are formed per ~6-min run, 97encapsulating thousands of cells in GEMs. Cells are loaded at a limiting dilution to minimize co- 98occurrence of multiple cells in the same GEM. 100Cell lysis begins immediately after encapsulation. Gel beads automatically dissolve to 101 release their oligonucleotides for reverse transcription of poly-adenylated RNAs. Each cDNA 102 molecule contains a UMI and shared barcode per GEM, and ends with a template switching 103 oligo at the 3' end (Fig. 1e). Next, the emulsion is broken and barcoded cDNA is pooled for 104PCR amplification, using primers complementary to the switch oligos and sequencing adapters. Methods, Fig. 1f). Briefly, 98-nt of Read1s were aligned against the union of human (hg19) 123peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/065912 doi: bioRxiv preprint first posted online 6 Based on the distribution of total UMI counts for each barcode (Online Methods), we 124 estimated that 1,012 GEMs contained cells, of which 482 and 538 contained reads that mapped 125 primarily to the human and mouse transcriptome, respectively (and will be referred to as human 126 and mouse GEMs) (Fig. 2a). >83% of UMI counts were associated with cell barcodes, 127indicating low background of cell-free RNA. Eight cell-containing GEMs had a substantial 128 fraction of human and mouse UMI counts (the UMI count is >1% of each species' UMI...
We present a simple and robust isotachophoresis (ITP) method that can be integrated with microchip-based capillary electrophoresis (CE) devices to achieve millionfold sample stacking. We performed an experimental parametric study to show the effects of initial sample ion concentration, leading ion concentration, and trailing ion concentration on ITP stacking. We also discuss the usefulness and limitations of a simple one-dimensional nondispersive model and a scaling analysis for dispersion rate. We found that a single-column ITP configuration together with electroosmotic flow suppression and high leading ion concentration provide high-performance ITP and can be integrated readily with CE separation. We demonstrated detection of trace of 100 fM Alexa Fluor 488 (signal-to-noise ratio of 11) with a concentration increase of a factor of 2 x 10(6). Application of our ITP/CE protocol to the stacking and separation of negatively charged fluorescent tracers (Alexa Fluor 488 and bodipy) resulted in a concentration increase of 6.4 x 10(4) and a signal increase of 4.5 x 10(5). The ITP/CE protocol can be performed with a standard microchannel cross design or simple flow control. The method can be implemented with available off-the-shelf chip systems using off-the-shelf voltage control systems and buffer chemistries.
Large-scale population based 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, standard short-read approaches, used primarily due to accuracy, throughput and costs, fail to give a complete picture of a genome. They struggle to identify large, balanced structural events, cannot access repetitive regions of the genome and fail to resolve the human genome into its two haplotypes. Here we describe an approach that retains long range information while harnessing the advantages of short reads. Starting from only~ ng of DNA, we produce barcoded short read libraries. The use of novel informatic approaches allows for the barcoded short reads to be associated with the long molecules of origin producing a novel datatype known as 'Linked-Reads'. This approach allows for simultaneous detection of small and large variants from a single Linked-Read library. We have previously demonstrated the utility of whole genome Linked-Reads (lrWGS) for performing diploid, de novo assembly of individual genomes (Weisenfeld et al. ). In this manuscript, weshow the advantages of Linked-Reads over standard short read approaches for reference based analysis. We demonstrate the ability of Linked-Reads to reconstruct megabase scale haplotypes and to recover parts of the genome that are typically inaccessible to short reads, including phenotypically important genes such as STRC, SMN and SMN . We demonstrate the ability of both lrWGS and Linked-Read Whole Exome Sequencing (lrWES) to identify complex structural variations, including balanced events, single exon deletions, and single exon duplications. The data presented here show that Linked-Reads provide a scalable approach for comprehensive genome analysis that is not possible using short reads alone.
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