Naveen Ramalingam

Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex

Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (∼50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In the developing cortex, we identify diverse cell types, including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells.

Real-time PCR microfluidic devices with concurrent electrochemical detection.

Electrochemistry-based detection methods hold great potential towards development of hand-held nucleic-acid analyses instruments. In this work, we demonstrate the implementation of in situ electrochemical (EC) detection method in a microfluidic flow-through EC-qPCR (FTEC-qPCR) device, where both the amplification of the target nucleic-acid sequence and subsequent EC detection of the PCR amplicon are realized simultaneously at selected PCR cycles in the same device. The FTEC-qPCR device utilizes methylene blue (MB), an electroactive DNA intercalator, for electrochemical signal measurements in the presence of PCR reagent components. Our EC detection method is advantageous, when compared to other existing EC methods for PCR amplicon analysis, since FTEC-qPCR does not require probe-modified electrodes, or asymmetric PCR, or solid-phase PCR. Key technical issues related to surface passivation, electrochemical measurement, PCR inhibition by metal electrode, bubble-free PCR, were investigated. By controlling the concentration of MB and the exposure of PCR mixture to the bare metal electrode, we successfully demonstrated electrochemical measurement of MB in solution-phase, symmetric PCR by amplifying a fragment of lambda phage DNA. The threshold cycle (C(t)) values for both the electrochemical and fluorescence-based assays decreased linearly with the increase of the input target quantity. The sensitivity of EC-based detection of PCR products is comparable to the sensitivity of an optical fluorescence detection system.

Micro air bubble formation and its control during polymerase chain reaction (PCR) in polydimethylsiloxane (PDMS) microreactors

Air bubble formation during polymerase chain reaction (PCR) thermocycling in microreactors has been reported as one of the major causes for PCR failure. In this paper we investigate the locations, mechanisms and other characteristics of the micro bubble formation inside a PCR microreactor array chip made by polydimethylsiloxane (PDMS) bonded with glass. The bubble formation is found to be strongly related to the micro features inside the microreactors and inside the chip bonding interface, especially near the inner corners of the microreactors, which are dependent on the micro-fabrication methods used. Gas permeability of PDMS and the wetting property of PCR sample also have influence on the air bubble formation. After investigation of various methods to control the bubble formation, we present the two most viable ones through micro bubble absorption and chip bonding interface modification. Finally, a bubble-free PCR in PDMS microreactors is demonstrated, in which the micro bubbles are suppressed with a bonding interface cladding technique.

Microfluidic devices harboring unsealed reactors for real-time isothermal helicase-dependent amplification

High-throughput microchip devices used for nucleic-acid amplification require sealed reactors. This is to prevent evaporative loss of the amplification mixture and cross-contamination, which may occur among fluidically connected reactors. In most high-throughput nucleic-acid amplification devices, reactor sealing is achieved by microvalves. Additionally, these devices require micropumps to distribute amplification mixture into an array of reactors, thereby increasing the device cost, and adding complexity to the chip fabrication and operation processes. To overcome these limitations, we report microfluidic devices harboring open (unsealed) reactors in conjunction with a single-step capillary based flow scheme for sequential distribution of amplification mixture into an array of reactors. Concern about evaporative loss in unsealed reactors have been addressed by optimized reactor design, smooth internal reactor surfaces, and incorporation of a localized heating scheme for the reactors, in which isothermal, real-time helicase-dependent amplification (HDA) was performed.

Real-time PCR array chip with capillary-driven sample loading and reactor sealing for point-of-care applications

A major challenge for the lab-on-a-chip (LOC) community is to develop point-of-care diagnostic chips that do not use instruments. Such instruments include pumping or liquid handling devices for distribution of patient’s nucleic-acid test sample among an array of reactors and microvalves or mechanical parts to seal these reactors. In this paper, we report the development of a primer pair pre-loaded PCR array chip, in which the loading of the PCR mixture into an array of reactors and subsequent sealing of the reactors were realized by a novel capillary-based microfluidics with a manual two-step pipetting operations. The chip is capable of performing simultaneous (parallel) analyses of multiple gene targets and its performance was tested by amplifying twelve different gene targets against cDNA template from human hepatocellular carcinoma using SYBR Green I fluorescent dye. The versatility and reproducibility of the PCR-array chip are demonstrated by real-time PCR amplification of the BNI-1 fragment of SARS cDNA cloned in a plasmid vector. The reactor-to-reactor diffusion of the pre-loaded primer pairs in the chip is investigated to eliminate the possibility of primer cross-contamination. Key technical issues such as PCR mixture loss in gas-permeable PDMS chip layer and bubble generation due to different PDMS-glass bonding methods are investigated.

Single-cell profiling approaches to probing tumor heterogeneity.

Tumor heterogeneity is a major hindrance in cancer classification, diagnosis and treatment. Recent technological advances have begun to reveal the true extent of its heterogeneity. Single‐cell analysis (SCA) is emerging as an important approach to detect variations in morphology, genetic or proteomic expression. In this review, we revisit the issue of inter‐ and intra‐tumor heterogeneity, and list various modes of SCA techniques (cell‐based, nucleic acid‐based, protein‐based, metabolite‐based and lipid‐based) presently used for cancer characterization. We further discuss the advantages of SCA over pooled cell analysis, as well as the limitations of conventional techniques. Emerging trends, such as high‐throughput sequencing, are also mentioned as improved means for cancer profiling. Collectively, these applications have the potential for breakthroughs in cancer treatment.

Fluidic Logic Used in a Systems Approach to Enable Integrated Single-Cell Functional Analysis

The study of single cells has evolved over the past several years to include expression and genomic analysis of an increasing number of single cells. Several studies have demonstrated wide spread variation and heterogeneity within cell populations of similar phenotype. While the characterization of these populations will likely set the foundation for our understanding of genomic- and expression-based diversity, it will not be able to link the functional differences of a single cell to its underlying genomic structure and activity. Currently, it is difficult to perturb single cells in a controlled environment, monitor and measure the response due to perturbation, and link these response measurements to downstream genomic and transcriptomic analysis. In order to address this challenge, we developed a platform to integrate and miniaturize many of the experimental steps required to study single-cell function. The heart of this platform is an elastomer-based integrated fluidic circuit that uses fluidic logic to select and sequester specific single cells based on a phenotypic trait for downstream experimentation. Experiments with sequestered cells that have been performed include on-chip culture, exposure to various stimulants, and post-exposure image-based response analysis, followed by preparation of the mRNA transcriptome for massively parallel sequencing analysis. The flexible system embodies experimental design and execution that enable routine functional studies of single cells.

Numerical and experimental study of capillary-driven flow of PCR solution in hybrid hydrophobic microfluidic networks.

Capillary-driven microfluidics is essential for development of point-of-care diagnostic micro-devices. Polymerase chain reaction (PCR)-based micro-devices are widely developed and used in such point-of-care settings. It is imperative to characterize the fluid parameters of PCR solution for designing efficient capillary-driven microfluidic networks. Generally, for numeric modelling, the fluid parameters of PCR solution are approximated to that of water. This procedure leads to inaccurate results, which are discrepant to experimental data. This paper describes mathematical modeling and experimental validation of capillary-driven flow inside Poly-(dimethyl) siloxane (PDMS)-glass hybrid micro-channels. Using experimentally measured PCR fluid parameters, the capillary meniscus displacement in PDMS-glass microfluidic ladder network is simulated using computational fluid dynamic (CFD), and experimentally verified to match with the simulated data.

Abstract 2923: Label-free enrichment and integrated full-length mRNA transcriptome analysis of single live circulating tumor cells from breast cancer patients

Background Label-free methods for isolating circulating tumor cells (CTCs) are attractive because they provide an opportunity to analyze a larger set of CTCs that may otherwise be missed due to variable or no expression of protein (label) markers. Understanding genetic and functional heterogeneity in CTCs allows us to gain insight into the mechanisms underscoring metastasis, drug resistance, and tumor aggressiveness. Currently, a simple workflow for isolation and molecular characterization of single CTCs by mRNA sequencing is lacking. In order to address this challenge, we developed a label-free workflow to isolate CTCs from breast cancer patients for full-length mRNA sequencing analysis by integrating the ClearCell® FX System with the Polaris™ system. The ClearCell FX system processes blood samples from cancer patients and enriches for CTCs in a label-free antibody-independent manner. The low level of nonspecifically isolated white blood cells from ClearCell FX is further depleted on the Polaris system by negative enrichment of viable CTCs. This unique integration of systems will enable researchers to perturb single CTCs in a controlled environment, monitor and measure the response due to perturbation, and link these response measurements to downstream genomic and transcriptomic analysis. Method and Results CTCs from 7.5 mL of peripheral blood sample from breast cancer patients were enriched using ClearCell FX. To differentiate larger blood cells from putative CTCs, we stained the enriched cells with Alexa Fluor® 647-conjugated CD45 and CD31 to identify leukocytes and endothelial cells, respectively. Calcein AM (live cell marker) and CellTracker™ Orange (universal cell marker) were added to identify live cells. Single CTCs were selected on Polaris (Fluidigm) system, lysed and reverse-transcribed, and cDNA were preamplified on the Polaris integrated fluidic circuit (IFC). Sequencing libraries were generated using the Nextera® kit and sequenced on Illumina® MiSeq™ and NextSeq™ systems. We successfully processed blood samples from four patients. Sequenced data showed high-quality metrics, with read depth of up to 2.5 million reads (MiSeq) or 60 million reads (NextSeq), with a low percentage of mapped reads to ribosomal RNA and mitochondrial RNA. Unsupervised hierarchical clustering of gene expression data showed clustering by patient, but considerable heterogeneity was also observed among the CTCs from the same patient. We will provide insights into full-length mRNA transcriptome of single CTCs from triple negative breast cancer patient. Conclusion We present the feasibility of integrating two microfluidics platforms to capture single CTCs for transcriptome and functional study. Our data suggests that the heterogeneity of tumor sample and characterization of metastatic processes can be elucidated from single-cell mRNA sequencing of CTCs. Citation Format: Naveen Ramalingam, Yi Fang Lee, Lukasz Szpankowski, Anne Leyrat, Brian Fowler, Jovina Tan, Chong Tracy Lu, Ninez Delos Angeles, Chad Sanada, Cassandra Greene, Kyle Hukari, Andrew Wu, Yoon-Sim Yap, Jay West, Ali Asgar Bhagat. Label-free enrichment and integrated full-length mRNA transcriptome analysis of single live circulating tumor cells from breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2923. doi:10.1158/1538-7445.AM2017-2923

Acetylated bovine serum albumin differentially inhibits polymerase chain reaction in microdevices

Bovine serum albumin (BSA) is widely used as an additive in polymerase chain reaction (PCR)-based microfluidic devices to passivate reactors and alleviate nucleic-acid amplification. BSA is available commercially in two types: either acetylated or non-acetylated. A survey of literature indicates that both types of BSA are used in PCR-based microfluidic devices. Our study results reveal that the use of acetylated BSA in PCR micro-devices leads to differential inhibition of PCR, compared to non-acetylated BSA. This result is noticed for the first time, and the differential inhibition generally goes un-noticed, as compared to complete PCR inhibition.