Single-cell DNA and RNA sequencing of circulating tumor cells

Masato Kojima,Takahiro Fukazawa,Eiso Hiyama,Shinya Takahashi,Isamu Saeki,Takanori Harada,Sho Kurihara

doi:10.1038/s41598-021-02165-7

Masato Kojima, Takahiro Fukazawa + Show 5 more

Open Access

https://doi.org/10.1038/s41598-021-02165-7

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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.

Highlights

Circulating tumor cells (CTCs) have been considered to represent the precise tumor heterogeneity and overall tumor c haracteristics[1]
TGW neuroblastoma cells were spiked into blood derived from a healthy volunteer for comparison of whole-genome amplifications (WGA) and whole-transcriptome amplifications (WTA)
It is known that PCR-based and PCR with multiple displacement amplification (MDA)-hybridized WGA methods generate high amplification uniformity, whereas the MDA-based WGA method shows high amplification efficiency but generates amplification bias through high processive amplification by phi[29] DNA polymerase, contributing to lower amplification uniformity[5]

Summary

Introduction

Circulating tumor cells (CTCs) have been considered to represent the precise tumor heterogeneity and overall tumor c haracteristics[1]. Similar to WGA, many WTA methods can be used for low-input RNA samples These methods use different priming strategies (polyT priming or random priming with ribosomal RNA depletion), second strand cDNA synthesis (polyA tailing or template switching), and cDNA amplification (PCR or isothermal in vitro transcription) approaches[6,7]. Variations in these methods can affect amplification efficiency and accuracy, as well as introduce unwanted bias. After isolating the CTCs by FACS, single-cell DNA and RNA were amplified by various WGA and WTA methods. We evaluated the performance of single-cell sequencing by next-generation sequencing (NGS)

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Results

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