Peilin Chen

Single cell RNA Seq reveals dynamic paracrine control of cellular variation

High-throughput single-cell transcriptomics offers an unbiased approach for understanding the extent, basis and function of gene expression variation between seemingly identical cells. Here we sequence single-cell RNA-seq libraries prepared from over 1,700 primary mouse bone-marrow-derived dendritic cells spanning several experimental conditions. We find substantial variation between identically stimulated dendritic cells, in both the fraction of cells detectably expressing a given messenger RNA and the transcript’s level within expressing cells. Distinct gene modules are characterized by different temporal heterogeneity profiles. In particular, a ‘core’ module of antiviral genes is expressed very early by a few ‘precocious’ cells in response to uniform stimulation with a pathogenic component, but is later activated in all cells. By stimulating cells individually in sealed microfluidic chambers, analysing dendritic cells from knockout mice, and modulating secretion and extracellular signalling, we show that this response is coordinated by interferon-mediated paracrine signalling from these precocious cells. Notably, preventing cell-to-cell communication also substantially reduces variability between cells in the expression of an early-induced ‘peaked’ inflammatory module, suggesting that paracrine signalling additionally represses part of the inflammatory program. Our study highlights the importance of cell-to-cell communication in controlling cellular heterogeneity and reveals general strategies that multicellular populations can use to establish complex dynamic responses.

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.

Abstract 1147: High-throughput nanofluidic PCR-based preparation of 454 sequencing libraries for identifying mutations in EGFR and MET

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor (MET) play important roles in cancer development and are promising targets for anticancer therapies. Mutations in the EGFR and MET genes are associated with multiple types of cancer and the ability to detect variants in these genes is crucial for a better understanding of disease progression and response to targeted therapeutics. Next-generation sequencers (e.g. 454, Illumina) hold great potential for detecting disease-specific alterations within patient populations for a given cancer type. However, current approaches for targeted resequencing of specific genetic regions are not well suited to studying the hundreds or thousands of patient samples required to assign significance to mutations. We have developed a novel nanofluidic platform, the Access Array™ system, which enables the robust, simultaneous amplification of 48 PCR products (amplicon) from 48 samples in parallel using only 50ng input DNA per sample. In addition, we have developed a primer design strategy that incorporates sample-specific barcodes and 454 sequencing adaptors into each amplicon, removing the need for library preparation before sequencing. We will present sequencing data collected from amplicon libraries prepared using the Access Array system to amplify 48 exon regions of the EGFR and MET genes from the NCI-60 cell lines. Sequencing data from these libraries demonstrates excellent uniformity in representation between amplicons and across samples. We will present data mapping mutations within these genes across the NCI-60, and discuss the use of Access Array enrichment panels in monitoring mutations across large patient populations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1147.

Abstract 1164: Parallel preparation of targeted resequencing libraries from 480 genomic regions using multiplex PCR on the Access Array system

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Next generation sequencing platforms have dramatically reduced sequencing costs. However, it currently remains too expensive to routinely resequence entire human genomes in order to discover genetic variants or somatic mutations underlying tumorigenesis. Therefore, a need exists for multiplexed, targeted amplification methods that allow for the analysis of multiple genomic regions in large cohorts. Available targeted enrichment technologies are either aimed at the capture of regions of interest from a single sample, exhibit uneven representation or require significant amounts of input material. The novel microfluidic platform, the Access Array system, combines 48 samples with 48 primer sets resulting in 2,304 simultaneously occurring PCR amplifications requiring as little as 50ng DNA per sample. PCR products generated on the Access Array system can be used for sequencing on all next-generation sequencing platforms, including 454 GS FLX and Illumina GAII. To increase coverage and throughput, PCR reactions can be multiplexed within Access Array chips generating up to 480 amplicons per sample. As proof-of-principal experiments, we have carried out multiplexed amplifications of a set of commonly mutated cancer gene exons across 48 genomic DNA samples. In initial experiments, 580 primer pairs were validated in individual PCR reactions in 96-well plates. Each primer pair was designed to include 5’ sequences that allow for the incorporation of 454 and Illumina adapters necessary for subsequent emPCR and cluster generation, respectively. 480 primer pairs that produced a single band of the correct size, as determined on a Caliper LabChip system, were selected for multiplex PCR experiments. Primer pairs yielding amplicons with a similar size were combined in groups of 10 sets, resulting in 48 primer pools of 10 primer pairs each. Multiplex PCR was carried out on Access Array chips, followed by harvesting of the 48 amplicon pools. Each pool was diluted and then subjected to a second round of PCR in standard 96-well plates with barcoded universal primers corresponding to the 454 and Illumina sequences. The resulting products are 48 uniquely barcoded amplicon pools, each comprising 480 amplicons derived from one sample, that are ready for sequencing. We will present sequencing data generated on both 454 and Illumina systems. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1164.