Full-length single-cell RNA-seq applied to a viral human cancer: applications to HPV expression and splicing analysis in HeLa S3 cells

Abstract

BackgroundViral infection causes multiple forms of human cancer, and HPV infection is the primary factor in cervical carcinomas. Recent single-cell RNA-seq studies highlight the tumor heterogeneity present in most cancers, but virally induced tumors have not been studied. HeLa is a well characterized HPV+ cervical cancer cell line.ResultWe developed a new high throughput platform to prepare single-cell RNA on a nanoliter scale based on a customized microwell chip. Using this method, we successfully amplified full-length transcripts of 669 single HeLa S3 cells and 40 of them were randomly selected to perform single-cell RNA sequencing. Based on these data, we obtained a comprehensive understanding of the heterogeneity of HeLa S3 cells in gene expression, alternative splicing and fusions. Furthermore, we identified a high diversity of HPV-18 expression and splicing at the single-cell level. By co-expression analysis we identified 283 E6, E7 co-regulated genes, including CDC25, PCNA, PLK4, BUB1B and IRF1 known to interact with HPV viral proteins.ConclusionOur results reveal the heterogeneity of a virus-infected cell line. It not only provides a transcriptome characterization of HeLa S3 cells at the single cell level, but is a demonstration of the power of single cell RNA-seq analysis of virally infected cells and cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s13742-015-0091-4) contains supplementary material, which is available to authorized users.

Human papillomavirus genome integration in squamous carcinogenesis: what have next‐generation sequencing studies taught us?

“…The use of into the distal host genome, producing a fusion transcript that can appear as a genuine breakpoint ( Figure 2). This, in part, has led to up to 15 HPV18 integration sites being claimed in HeLa cells [17,18], whereas only four are usually confirmed [11,13,19]. The need for verification of NGS findings by Sanger sequencing applies to both RNA-seq and DNA-seq.…”

Evolution of multiple cell clones over a 29-year period of a CLL patient

“…To investigate gene expression profiles over time, we performed single-cell RNA transcriptome sequencing (RNA-seq)13 on 300 unsorted tumour cells from the year of year 10, 20, 23, 26 and 28 (Supplementary Fig. 13; Supplementary Table 4).…”

“…All complementary DNA products of single CLL B cells in this study were prepared by a microwell based a platform called MIRALCS13. A total of 1 ng complementary DNA product from each cell was used for library construction using the TruePrepTM Mini DNA Sample Prep Kit (Vazyme Biotech) followed by barcode labelling.…”

“…The fragments per kilobase of exon per million fragments mapped (FPKM) values were calculated using the R package edgeR47. The detailed process and parameters were described in a recent publication13. The single cells with less than one million reads mapping to gene regions were removed from further analysis.…”

Detection of HPV E7 Transcription at Single-Cell Resolution in Epidermis

“…To date, the application of single-cell technology for viral detection and viral genomics has been limited to low-throughput and plate-based methods, including single-cell genome and/or RNA sequencing to detect HIV DNA/RNA in isolated peripheral blood mononucleated single cells, (Josefsson et al, 2013, Wiegand et al, 2017, HPV18 in 40 HeLa S3 cells using a plate-based, modified SMART-seq2 protocol (Wu et al, 2015), and Hepatitis C virus using plate-based single-cell qPCR (McWilliam Leitch and McLauchlan, 2013).…”