Single cell transcriptome analysis of MCF-7 reveals consistently and inconsistently expressed gene groups each associated with distinct cellular localization and functions

<div><p>Single cell transcriptome (SCT) analysis provides superior resolution to illustrate tumor cell heterogeneity for clinical implications. We characterized four SCTs of MCF-7 using 143 housekeeping genes (HKGs) as control, of which lactate dehydrogenase B (LDHB) expression is silenced. These SCT libraries mapped to 11,423, 11,486, 10,380, and 11,306 RefSeq genes (UCSC), respectively. High consistency in HKG expression levels across all four SCTs, along with transcriptional silencing of <i>LDHB</i>, was observed, suggesting a high sensitivity and reproducibility of the SCT analysis. Cross-library comparison on expression levels by scatter plotting revealed a linear correlation and an 83–94% overlap in transcript isoforms and expressed genes were also observed. To gain insight of transcriptional diversity among the SCTs, expressed genes were split into consistently expressed (CE) (expressed in all SCTs) and inconsistently expressed (IE) (expressed in some but not all SCTs) genes for further characterization, along with the 142 expressed HKGs as a reference. Distinct transcriptional strengths were found among these groups, with averages of 1,612.0, 88.0 and 1.2 FPKM for HKGs, CE and IE, respectively. Comparison between CE and IE groups further indicated that expressions of CE genes vary more significantly than that of IE genes. Gene Ontology analysis indicated that proteins encoded by CE genes are mainly involved in fundamental intracellular activities, while proteins encoded by IE genes are mainly for extracellular activities, especially acting as receptors or ion channels. The diversified gene expressions, especially for those encoded by IE genes, may contribute to cancer drug resistance.</p></div

An example of single cell transcriptome profiles produced by Galaxy.

<p>Genome-wide RNA-Seq shotgun sequences of sC4, sC5, sC6 and sC8 single cell transcriptomes (highlighted in different colors) were analyzed by Galaxy pipeline which adopts Tophat for mapping (against hg19) and Cufflinks for transcript isoform identification. The height of each cluster represents its relative expression level. Some peaks are not shown completely due to space constraint. Introns (thin grey lines) between exons and intergenic regions (thick grey lines) are barely seen between exons.</p

Expression level distributions for the 142 expressed reference HKGs, CE group, and IE group.

<p>For every gene in every group, the expression levels in all SCTs were averaged and sorted. FPKM values at 5%, 15%, 25%, 35%, 45%, 55%, 65%, 75%, 85% and 95% were sampled and converted by log₁₀ and plotted for cross-library comparison.</p

Expression of the 143 reference housekeeping genes.

<p>Expression of the 143 reference housekeeping genes.</p

Comparison of coefficient of variation for HKGs, CE and IE genes.

<p>CVs were calculated for all three gene groups, including housekeeping genes, consistently expressed genes, and inconsistently expressed genes and displayed separately for all single-cell library.</p

GO cellular component analysis for the consistently expressed (CE) genes.

<p>Genes consistently expressed in all four single cell transcriptome libraries are analyzed using Gene Ontology cellular component database. Components associated with major terms are highlighted by color, e.g., ‘organelle’ in green, ‘intracellular’ in red, and ‘mitochondrion’-related in purple.</p

Distribution of coefficient of variation (CV) of the 143 reference HKGs.

<p>The coefficient of variation was calculated for the expressions of each 143 reference HKGs in four SCT libraries and then plotted against their corresponding percentage. As shown in the figure, about 80% of the CVs were less than 0.55.</p

Scatter plots to show the correlation in gene expression levels for all two-library combinations of all four SCT libraries.

<p>The gene expression levels, in FPKM, of all expressed genes are plotted for any two-library combination of the SCT libraries.</p