气道抽吸液标本单细胞转录组测序前细胞处理技术的比较

Comparison of pretreatment techniques for single-cell RNA sequencing of airway aspirate specimens

目的探索适用于气道抽吸液标本单细胞转录组测序的细胞前处理方法。方法从4例急性呼吸道感染患者中新鲜采集4份气道抽吸液标本, 用痰消化液消化后过细胞滤网制备成单细胞悬液, 分别用新鲜细胞直接建库(direct emulsion, DE)、用10×Genomics的单细胞RNA固定试剂固定后混合建库(direct-fix, DF)、将细胞冻存复苏再固定后(frozen-fix, FF)混合建库3种方法操作, 3种方法均以104个细胞进行油包水处理, 用10×Genomics平台进行单细胞测序, 对获取的细胞数量、数据质量、注释的细胞种类、标记基因的表达水平进行比较分析, 用Pearson相关性分析3种前处理方法获得的同种细胞亚群高变基因表达差异。比较3种前处理方法获得的同一细胞亚群的差异基因在不同方法之间的表达水平。通过Pearson相关分析研究3种前处理方法获得的同一细胞亚群之间差异基因表达的相关性。结果 DE、FF、DF方法获得的单细胞数量中位数分别为2 733、1 140和5 897个(P>0.05), 唯一分子识别符高于500, 基因中位数分别为801、887、1 259(P>0.05)。评估RNA复杂度的新颖性评分超过0.8的细胞比例分别为99%、87%和93%。3种方法获得的细胞亚群均有鳞状上皮细胞、分泌细胞、纤毛细胞、T淋巴细胞、B淋巴细胞、巨噬细胞、浆细胞、中性粒细胞等9种细胞亚群, 与DE方法比较, DF和FF方法可多获得特异性高表达角蛋白5的基底细胞。3种前处理方法中同一细胞亚群差异基因和高变基因在不同方法之间表达高度一致, 具有显著相关性(P<0.001)。结论在相同的测序数据量下, 气道抽吸液标本单细胞悬液先固定再进行探针杂交和转录组测序的方法得到的数据质量与新鲜细胞直接操作的质量相近, 该方法更适用于临床样本的单细胞转录组测序分析的样本前处理过程。

Objective To investigate the appropriate pretreatment methods for single cell RNA sequencing of airway aspirate cells.Methods Four fresh airway aspirate specimens were collected from four patients with acute respiratory tract infections.These specimens were digested with airway aspirate digester and prepared into single cell suspension.The cells were used for library construction directly(DE),or fixed with 10×Genomics Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit and then mixed to construct the library(DF),or cryopreserved,thawed,fixed(FF)before mixed to construct the library.All three methods were treated with oil emulsion using 104 cells and subjected to single-cell sequencing using the 10×Genomics platform.The number of obtained cells,data quality,annotated cell types and expression of marker genes were analyzed.Differences in the expression of highly variable genes(HVGs)of the same cell subsets obtained by the three pretreatment methods were compared using Pearson correlation.Expression of the differentially expressed genes in the same cell subpopulation obtained by different pretreatment methods was also compared.The correlation of the expression of differentially expressed genes between the same cell subsets obtained by the three pretreatment methods was analyzed by Pearson correlation.Results The median numbers of single cells obtained using DE,FF and DF methods were 2733,1140 and 5897(P>0.05).The unique molecular identifiers were higher than 500.The median numbers of genes obtained using the three methods were 801,887 and 1259(P>0.05).The cells with novelty score over 0.8 accounted for 99%,87%and 93%,respectively.There were nine cell subsets obtained by the three methods,including squamous cells,secretory cells,ciliated cells,T cells,B cells,macrophages,plasma cells and neutrophils.DF and FF methods could obtain more basal cells with specific high expression of keratin 5 than DE method.The differentially expressed and highly variable genes in the same cell subsets obtained by the three pretreatment methods showed high consistency in their expression with a significant correlation(P<0.001).Conclusions Under the same sequencing data volume,the quality of data obtained from fixed airway aspirate single-cell suspensions using the method of probe hybridization and transcriptome sequencing was comparable to that obtained directly from fresh cells.This method was more suitable for the pretreatment of clinical samples used for single-cell RNA sequencing.