Feasibility of a workflow for the molecular characterization of single cells by next generation sequencing.
Feasibility of a workflow for the molecular characterization of single cells by next generation sequencing.
复制标题
DOI：
10.1016/j.bdq.2015.07.002
复制
发表时间：
2015-09
影响因子：
--
通讯作者：
Pinzani P
中科院分区：
文献类型：
--
作者：
Salvianti F;Rotunno G;Galardi F;De Luca F;Pestrin M;Vannucchi AM;Di Leo A;Pazzagli M;Pinzani P
该研究的目的是探索使用单细胞下一代测序（NGS）方法从癌症患者中分离和分子表征单个循环肿瘤细胞（CTC）的方案的可行性。为了实现这一目标，我们使用通过将乳腺癌细胞系（MDA-MB-231）掺入健康供体的血液中获得的人工样品作为模型。通过CellSearch®富集并计数肿瘤细胞，随后通过DEPArray™分离以获得单个或合并的纯样品，以提交用于分析癌症中涉及的多个基因的突变状态。在全基因组扩增后，使用Ion AmpliSeq™ Cancer Hotspot Panel v2（Life Technologies）在Ion Torrent PGM™系统（Life Technologies）上通过NGS分析样品，所述Ion AmpliSeq™ Cancer Hotspot Panel v2（Life Technologies）设计用于研究50种癌基因和肿瘤抑制基因的基因组“热点”区域。我们成功地测序了5个单细胞，5个细胞的池和来自相同细胞系的细胞沉淀的DNA，测序反应的平均深度范围为1581至3479个读数。我们在18个基因中发现了27个序列变异，其中15个已经在COSMIC或dbSNP数据库中报道。我们证实了在BRAF和TP 53基因中存在两个体细胞突变，这已经在该细胞系中报道过，但也发现了新的突变和单核苷酸多态性。所有分析的样品共有三种变体，而18种仅存在于单个细胞中，表明同一细胞系内的高度异质性。本文提出了一个优化的工作流程，在单细胞中的多个基因的分子特征的NGS。所描述的管道可以容易地转移到来自肿瘤患者的单个CTC的研究。
The purpose of the study was to explore the feasibility of a protocol for the isolation and molecular characterization of single circulating tumor cells (CTCs) from cancer patients using a single-cell next generation sequencing (NGS) approach. To reach this goal we used as a model an artificial sample obtained by spiking a breast cancer cell line (MDA-MB-231) into the blood of a healthy donor. Tumor cells were enriched and enumerated by CellSearch® and subsequently isolated by DEPArray™ to obtain single or pooled pure samples to be submitted to the analysis of the mutational status of multiple genes involved in cancer. Upon whole genome amplification, samples were analysed by NGS on the Ion Torrent PGM™ system (Life Technologies) using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Life Technologies), designed to investigate genomic “hot spot” regions of 50 oncogenes and tumor suppressor genes. We successfully sequenced five single cells, a pool of 5 cells and DNA from a cellular pellet of the same cell line with a mean depth of the sequencing reaction ranging from 1581 to 3479 reads. We found 27 sequence variants in 18 genes, 15 of which already reported in the COSMIC or dbSNP databases. We confirmed the presence of two somatic mutations, in the BRAF and TP53 gene, which had been already reported for this cells line, but also found new mutations and single nucleotide polymorphisms. Three variants were common to all the analysed samples, while 18 were present only in a single cell suggesting a high heterogeneity within the same cell line. This paper presents an optimized workflow for the molecular characterization of multiple genes in single cells by NGS. The described pipeline can be easily transferred to the study of single CTCs from oncologic patients.