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Benchmarking full-length transcript single cell mRNA sequencing protocols

ABSTRACT: Here we present a performance evaluation of three different plate-based scRNA-seq protocols. Two commercially available kits; NEBNext Single Cell/ Low Input RNA Library Prep Kit (NEB), SMART-seq HT kit (Takara), and non-commercially available protocol Genome & transcriptome sequencing (G&T). We evaluated each kit based upon sensitivity, reproducibility, ease of use and price point per cell. This evaluation is specifically relevant for implementation of scRNA-seq in e.g. diagnostics, requiring high sensitivity in regards of gene detection, high reproducibility between samples, minimum hands on time and smooth sample preparation for technicians, as well as keeping a reasonable price point per patient. G&T protocol delivered the highest detection of genes per single cell, at the absolute lowest price point. Takara's kit delivered likewise high gene detection per single cell, and high reproducibility between sample, however at the absolute highest price points. Here we present pros and cons of each protocol, sequencing breast cancer cell line T47D.

ORGANISM(S): Homo sapiens

PROVIDER: GSE155506 | GEO | 2020/08/01

REPOSITORIES: GEO

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Project description:The accurate mapping of recurring DNA copy number aberrations (CNAs), a hallmark feature of the cancer genome, has facilitated the discovery of tumor suppressor genes and oncogenes. Microarray-based assays designed to detect these chromosomal copy number alterations on a genome-wide and high-resolution scale have emerged as a cornerstone technology in the genomic era. The diversity of commercially-available platforms prompted a systematic comparison of five copy number profiling assays for their ability to detect 2-fold copy number gain and loss (4n or 1n, respectively) as well as focal high-amplitude CNAs. Here, using a collection of established human melanoma cell lines, we defined the reproducibility, absolute signals, signal:noise, false-positive and false-negative rates for each of the five assays against ground-truth defined by Spectral Karyotyping (SKY), in addition to comparing the concordance of CNAs detection by two high-resolution Agilent and Affymetrix microarray platforms. Our analyses concluded that the Agilentâs 60mer oligo-microarray with probe design optimized for genomic hybridization offers the highest sensitivity and specificity [area under Receiver Operator Characteristic (ROC) curve >0.99] while Affymetrixâs SNP microarray appears to offer better detection of CNAs in gene-poor region. Availability of these comparison results should guide study design decisions and facilitate further computational development. Keywords: comparative genomic hybridization We generated copy number profiles of a defined set of tumor cell lines on five oligo-microarray-based assays of three platforms (Agilent, Affymetrix and NimbleGen) and determined the reproducibility, signal and noise as well as sensitivity and specificity of each in detecting two-fold signals based on SKY-defined aberrations as ground-truth for comparison. In addition, high-density microarray assays from Agilent and Affymetrix platforms were further compared for definition of CNAs in an independent dataset using published analytical approaches. All arrays were run from the same DNA harvest from respective cell lines. Each array was run according to the protocol of the manufacturer.

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Benchmarking full-length transcript single cell mRNA sequencing protocols

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