Dataset Information

Cloud accelerated alignment and assembly of full-length single-cell RNA-seq data using Falco.

ABSTRACT: BACKGROUND:Read alignment and transcript assembly are the core of RNA-seq analysis for transcript isoform discovery. Nonetheless, current tools are not designed to be scalable for analysis of full-length bulk or single cell RNA-seq (scRNA-seq) data. The previous version of our cloud-based tool Falco only focuses on RNA-seq read counting, but does not allow for more flexible steps such as alignment and read assembly. RESULTS:The Falco framework can harness the parallel and distributed computing environment in modern cloud platforms to accelerate read alignment and transcript assembly of full-length bulk RNA-seq and scRNA-seq data. There are two new modes in Falco: alignment-only and transcript assembly. In the alignment-only mode, Falco can speed up the alignment process by 2.5-16.4x based on two public scRNA-seq datasets when compared to alignment on a highly optimised standalone computer. Furthermore, it also provides a 10x average speed-up compared to alignment using published cloud-enabled tool for read alignment, Rail-RNA. In the transcript assembly mode, Falco can speed up the transcript assembly process by 1.7-16.5x compared to performing transcript assembly on a highly optimised computer. CONCLUSION:Falco is a significantly updated open source big data processing framework that enables scalable and accelerated alignment and assembly of full-length scRNA-seq data on the cloud. The source code can be found at https://github.com/VCCRI/Falco.

SUBMITTER: Yang A

PROVIDER: S-EPMC6936136 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Publications

Cloud accelerated alignment and assembly of full-length single-cell RNA-seq data using Falco.

Yang Andrian A Kishore Abhinav A Phipps Benjamin B Ho Joshua W K JWK

BMC genomics 20191230 Suppl 10

<h4>Background</h4>Read alignment and transcript assembly are the core of RNA-seq analysis for transcript isoform discovery. Nonetheless, current tools are not designed to be scalable for analysis of full-length bulk or single cell RNA-seq (scRNA-seq) data. The previous version of our cloud-based tool Falco only focuses on RNA-seq read counting, but does not allow for more flexible steps such as alignment and read assembly.<h4>Results</h4>The Falco framework can harness the parallel and distribu ...[more]

PMID: 31888474

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Project description:Single-cell characterization techniques, such as mRNA-seq, have been applied to a diverse range of applications in cancer biology, yielding great insight into mechanisms leading to therapy resistance and tumor clonality. While single-cell techniques can yield a wealth of information, a common bottleneck is the lack of throughput, with many current processing methods being limited to the analysis of small volumes of single cell suspensions with cell densities on the order of 107 per mL. In this work, we present a high-throughput full-length mRNA-seq protocol incorporating a magnetic sifter and magnetic nanoparticle-antibody conjugates for rare cell enrichment, and Smart-seq2 chemistry for sequencing. We evaluate the efficiency and quality of this protocol with a simulated circulating tumor cell system, whereby non-small-cell lung cancer cell lines (NCI-H1650 and NCI-H1975) are spiked into whole blood, before being enriched for single-cell mRNA-seq by EpCAM-functionalized magnetic nanoparticles and the magnetic sifter. We obtain high efficiency (> 90%) capture and release of these simulated rare cells via the magnetic sifter, with reproducible transcriptome data. In addition, while mRNA-seq data is typically only used for gene expression analysis of transcriptomic data, we demonstrate the use of full-length mRNA-seq chemistries like Smart-seq2 to facilitate variant analysis of expressed genes. This enables the use of mRNA-seq data for differentiating cells in a heterogeneous population by both their phenotypic and variant profile. In a simulated heterogeneous mixture of circulating tumor cells in whole blood, we utilize this high-throughput protocol to differentiate these heterogeneous cells by both their phenotype (lung cancer versus white blood cells), and mutational profile (H1650 versus H1975 cells), in a single sequencing run. This high-throughput method can help facilitate single-cell analysis of rare cell populations, such as circulating tumor or endothelial cells, with demonstrably high-quality transcriptomic data.

Dataset Information

Cloud accelerated alignment and assembly of full-length single-cell RNA-seq data using Falco.

Publications

Cloud accelerated alignment and assembly of full-length single-cell RNA-seq data using Falco.

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