9 research outputs found
Recommended from our members
Next Generation Spatial Transcriptomics
Cells encode copies of genomic information in RNA molecules and traffic them to distinct subcellular destinations. These RNA molecules serve a milieu of functions including encoding protein sequences for local translation, regulating cellular functions, or aiding in intercellular communication. The breadth of subcellular RNA functionality is not well understood, and our capability of scaling our knowledge of spatial RNA biology to the entire transcriptome has, until now, been an inconceivable undertaking.In this dissertation, I describe our work developing a suite of software tools and novel spatial genomic methodologies, creating a unified vertically integrated stack of next-generation spatial transcriptomic technology. We developed RNAforest, RNAflux, and RNAcoloc, three first-in-class and best-in-class software algorithms for the statistical spatial analysis of RNA subcellular localization, wrapping them into a toolkit called Bento. Each of these tools enable different approaches to RNA distribution analysis, including supervised and unsupervised subcellular RNA patterning recognition, and compartment specific gene-gene pairwise colocalization. We demonstrated the utility of Bento by discovering organelle-specific RNA enrichment modulation by the chemotherapeutic Doxorubicin in Cardiomyocytes. We further utilized our philosophy of using RNA distribution to compute on cellular space by training a generative pix2pix-HD GAN model and a CellPose cyto2torch segmentation model to create a best-in-class cell segmentation model, RNA-masala, that computes cell boundaries in 3D accurately from RNA coordinates alone. RNA-masala is highly performant, leveraging GPU tensor computation making it feasible to segment single cells in 3D from large tissue spatial transcriptomic datasets such as a gigapixel whole mouse pup Xenium dataset.
Finally, we developed SPACE-seq, a next-generation spatial transcriptomic method that achieves a Moore’s Law scale inflection point in plexity (untargeted whole-transcriptome, isoform resolution), cost, as well as data dimensionality combining single-molecule imaging with single-molecule sequencing. SPACE-seq allows the measurement of the location and sequence of every unique molecule in every single cell in a tissue section, unlocking multi-scale RNA biology. Altogether, Bento, RNA-masala, and SPACE-seq take spatial transcriptomics takes RNA biology to new heights and introduces the possibility for creating digitized models of cell biology
Operation of Droplet-Microfluidic Devices with a Lab Centrifuge
Microfluidic devices are valuable for a variety of biotechnology applications, such as synthesizing biochemical libraries, screening enzymes, and analyzing single cells. However, normally, the devices are controlled using specialized pumps, which require expert knowledge to operate. Here, we demonstrate operation of poly(dimethylsiloxane) devices without pumps. We build a scaffold that holds the device and reagents to be infused in a format that can be inserted into a 50 mL falcon tube and spun in a common lab centrifuge. By controlling the device design and centrifuge spin speed, we infuse the reagents at controlled flow rates. We demonstrate the encapsulation and culture of clonal colonies of red and green Escherichia coli in droplets seeded from single cells
Operation of Droplet-Microfluidic Devices with a Lab Centrifuge
Microfluidic devices are valuable for a variety of biotechnology applications, such as synthesizing biochemical libraries, screening enzymes, and analyzing single cells. However, normally, the devices are controlled using specialized pumps, which require expert knowledge to operate. Here, we demonstrate operation of poly(dimethylsiloxane) devices without pumps. We build a scaffold that holds the device and reagents to be infused in a format that can be inserted into a 50 mL falcon tube and spun in a common lab centrifuge. By controlling the device design and centrifuge spin speed, we infuse the reagents at controlled flow rates. We demonstrate the encapsulation and culture of clonal colonies of red and green Escherichia coli in droplets seeded from single cells
Recommended from our members
Operation of Droplet-Microfluidic Devices with a Lab Centrifuge.
Microfluidic devices are valuable for a variety of biotechnology applications, such as synthesizing biochemical libraries, screening enzymes, and analyzing single cells. However, normally, the devices are controlled using specialized pumps, which require expert knowledge to operate. Here, we demonstrate operation of poly(dimethylsiloxane) devices without pumps. We build a scaffold that holds the device and reagents to be infused in a format that can be inserted into a 50 mL falcon tube and spun in a common lab centrifuge. By controlling the device design and centrifuge spin speed, we infuse the reagents at controlled flow rates. We demonstrate the encapsulation and culture of clonal colonies of red and green Escherichia coli in droplets seeded from single cells
Operation of Droplet-Microfluidic Devices with a Lab Centrifuge
Microfluidic devices are valuable for a variety of biotechnology applications, such as synthesizing biochemical libraries, screening enzymes, and analyzing single cells. However, normally, the devices are controlled using specialized pumps, which require expert knowledge to operate. Here, we demonstrate operation of poly(dimethylsiloxane) devices without pumps. We build a scaffold that holds the device and reagents to be infused in a format that can be inserted into a 50 mL falcon tube and spun in a common lab centrifuge. By controlling the device design and centrifuge spin speed, we infuse the reagents at controlled flow rates. We demonstrate the encapsulation and culture of clonal colonies of red and green Escherichia coli in droplets seeded from single cells
Recommended from our members
Single-cell genome sequencing at ultra-high-throughput with microfluidic droplet barcoding.
The application of single-cell genome sequencing to large cell populations has been hindered by technical challenges in isolating single cells during genome preparation. Here we present single-cell genomic sequencing (SiC-seq), which uses droplet microfluidics to isolate, fragment, and barcode the genomes of single cells, followed by Illumina sequencing of pooled DNA. We demonstrate ultra-high-throughput sequencing of >50,000 cells per run in a synthetic community of Gram-negative and Gram-positive bacteria and fungi. The sequenced genomes can be sorted in silico based on characteristic sequences. We use this approach to analyze the distributions of antibiotic-resistance genes, virulence factors, and phage sequences in microbial communities from an environmental sample. The ability to routinely sequence large populations of single cells will enable the de-convolution of genetic heterogeneity in diverse cell populations
Recommended from our members
Bulk double emulsification for flow cytometric analysis of microfluidic droplets
Droplet microfluidics is valuable for applications in chemistry and biology, but generates massive numbers of droplets that must be analyzed and sorted. Here, we describe a simple approach to bulk double emulsify microfluidic emulsions for analysis and sorting with commercial flow cytometers. We illustrate the method by using it to identify droplets based on nucleic acid content. Though simple, our method provides a general approach for analyzing and sorting microfluidic droplets without custom microfluidic double emulsifiers or sorters
Junior scientists spotlight social bonds in seminars for diversity, equity, and inclusion in STEM.
Disparities for women and minorities in science, technology, engineering, and math (STEM) careers have continued even amidst mounting evidence for the superior performance of diverse workforces. In response, we launched the Diversity and Science Lecture series, a cross-institutional platform where junior life scientists present their research and comment on diversity, equity, and inclusion in STEM. We characterize speaker representation from 79 profiles and investigate topic noteworthiness via quantitative content analysis of talk transcripts. Nearly every speaker discussed interpersonal support, and three-fifths of speakers commented on race or ethnicity. Other topics, such as sexual and gender minority identity, were less frequently addressed but highly salient to the speakers who mentioned them. We found that significantly co-occurring topics reflected not only conceptual similarity, such as terms for racial identities, but also intersectional significance, such as identifying as a Latina/Hispanic woman or Asian immigrant, and interactions between concerns and identities, including the heightened value of friendship to the LGBTQ community, which we reproduce using transcripts from an independent seminar series. Our approach to scholar profiles and talk transcripts serves as an example for transmuting hundreds of hours of scholarly discourse into rich datasets that can power computational audits of speaker diversity and illuminate speakers' personal and professional priorities
Recommended from our members
Automated, miniaturized, and scalable screening of healthcare workers, first responders, and students for SARS-CoV-2 in San Diego County
Background Successful containment strategies for SARS-CoV-2, the causative virus of the COVID-19 pandemic, have involved widespread population testing that identifies infections early and enables rapid contact tracing. In this study, we developed a rapid and inexpensive RT- qPCR testing pipeline for population-level SARS-CoV-2 detection, and used this pipeline to establish a clinical laboratory dedicated to COVID-19 testing at the University of California San Diego (UCSD) with a processing capacity of 6,000 samples per day and next-day result turnaround times. Methods and findings Using this pipeline, we screened 6,786 healthcare workers and first responders, and 21,220 students, faculty, and staff from UCSD. Additionally, we screened 6,031 preschool-grade 12 students and staff from public and private schools across San Diego County that remained fully or partially open for in-person teaching during the pandemic. Between April 17, 2020 and February 5, 2021, participants provided 161,582 nasal swabs that were tested for the presence of SARS-CoV-2. Overall, 752 positive tests were obtained, yielding a test positivity rate of 0.47%. While the presence of symptoms was significantly correlated with higher viral load, most of the COVID-19 positive participants who participated in symptom surveys were asymptomatic at the time of testing. The positivity rate among preschool-grade 12 schools that remained open for in-person teaching was similar to the positivity rate at UCSD and lower than that of San Diego County, with the children in private schools being less likely to test positive than the adults at these schools. Conclusions Most schools across the United States have been closed for in-person learning for much of the 2020-2021 school year, and their safe reopening is a national priority. However, as there are no vaccines against SARS-CoV-2 currently available to the majority of school-aged children, the traditional strategies of mandatory masking, physical distancing, and repeated viral testing of students and staff remain key components of risk mitigation in these settings. The data presented here suggest that the safety measures and repeated testing actions taken by participating healthcare and educational facilities were effective in preventing outbreaks, and that a similar combination of risk-mitigation strategies and repeated testing may be successfully adopted by other healthcare and educational systems