Single-cell DNA and RNA sequencing of circulating tumor cells

Abstract

Single-cell sequencing of circulating tumor cells can precisely represent tumor heterogeneity and provide useful information for cancer treatment and research. After spiking TGW neuroblastoma cells into blood derived from healthy volunteer, the cells were isolated by fluorescence-activated cell sorting. DNA and mRNA were amplified by four different whole-genome amplifications (WGA) and three whole-transcriptome amplifications (WTA) methods, followed by single-cell DNA and RNA sequencing. Multiple displacement amplification (MDA)-based WGA methods showed higher amplification efficiency than other methods with a comparable depth of coverage as the bulk sample. The uniformity of coverage greatly differed among samples (12.5–89.2%), with some samples evaluated by the MDA-based WGA method using phi29 DNA polymerase and random primers showing a high (> 80%) uniformity of coverage. The MDA-based WTA method less effectively amplified mRNA and showed non-specific gene expression patterns. The PCR-based WTA using template switching with locked nucleic acid technology accurately amplified mRNA from a single cell. Taken together, our results present a more reliable and adaptable approach for CTC profiling at the single-cell level. Such molecular information on CTCs derived from clinical patients will promote cancer treatment and research.

Single‐cell next‐generation sequencing of circulating tumor cells in patients with neuroblastoma

“…Quality check (QC) was performed using multiplex qPCR for eight cancer‐related genes: BRAF , EGFR , KIT , KRAS , NRAS , PIK3CA , PTEN , and TP53 . The QC standard was based on the number of threshold cycles (Ct) ≤ 20 obtained for these genes based on our previous study 7 …”

“…We isolated single neuroblastoma cells from biopsied tumor tissue (primary tumor cells: PTCs) from five patients with neuroblastoma (cases 6–10), to compare their gene expression profile with that of single CTCs. After dissociation of tissue specimen using a cell strainer, GD2 + CD90 + CD45 − CD235a − DAPI − cells were isolated using FACS as previously described 7 . In total, mRNA from 30 single PTCs and 48 single CTCs was amplified; specifically, 5, 10, 5, 5 and 5 amplified mRNA datasets for single PTCs, and 8, 10, 5, 5 and 20 amplified mRNA datasets for single CTCs were obtained from cases 6, 7, 8, 9, and 10, respectively, using SMART‐Seq HT Kit (TaKaRa Bio, Shiga, Japan).…”

“…The RNA collected from PTCs and CTCs from five patients with neuroblastoma (cases 6–10) was sequenced at single‐cell level as previously described 7 . Processing of FASTQ files and alignment of sequenced reads were performed in accordance with our previous study 7 .…”

“…The RNA collected from PTCs and CTCs from five patients with neuroblastoma (cases 6–10) was sequenced at single‐cell level as previously described 7 . Processing of FASTQ files and alignment of sequenced reads were performed in accordance with our previous study 7 . In total, 21 single PTCs and 42 single CTCs with over 500,000 reads from transcriptome were selected for data analysis; specifically, 3, 3, 5, 5, and 5 mRNA datasets for single PTCs, and 6, 6, 5, 5, and 20 mRNA datasets for single CTCs were analyzed from cases 6, 7, 8, 9, and 10, respectively.…”

“…In total, 21 single PTCs and 42 single CTCs with over 500,000 reads from transcriptome were selected for data analysis; specifically, 3, 3, 5, 5, and 5 mRNA datasets for single PTCs, and 6, 6, 5, 5, and 20 mRNA datasets for single CTCs were analyzed from cases 6, 7, 8, 9, and 10, respectively. The quantification of reads from transcripts and their normalization between samples were performed in accordance with our previous study 7 . Gene expression profiles were compared between PTCs and CTCs to identify differentially expressed genes (DEGs).…”