SCIFER: approach for analysis of LINE-1 mRNA expression in single cells at a single locus resolution.
SCIFER: approach for analysis of LINE-1 mRNA expression in single cells at a single locus resolution.
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DOI：
10.1186/s13100-022-00276-0
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发表时间：
2022-08-26
期刊：
影响因子：
4.9
通讯作者：
中科院分区：
文献类型：
--
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L1 mRNA的内源性表达是L1启动的突变事件的第一步。然而，个别细胞类型的贡献器官特异性L1 mRNA表达的模式仍然知之甚少，特别是在单位点分辨率。我们介绍了一种方法来量化表达的移动的元素在scRNA-Seq数据集的单位点分辨率称为单细胞实现找到表达反转录转座子（SCIFER）。SCIFER将scRNA-Seq读数与基因组进行唯一比对，并通过细胞特异性条形码从单细胞中提取比对结果。与使用默认参数进行的比对相比，该比对策略通过仅保留唯一映射到单个L1基因座的读段来提高L1基因座鉴定的准确性。使用经手动验证在由相同细胞系或器官生成的批量RNA-Seq数据集中表达的L1基因座列表明确鉴定在单细胞中表达的L1基因座。使用MCF 7细胞的SCIFER验证确定了单细胞中L1表达的最佳检测所需的技术参数。我们发现，在单细胞中L1表达的无监督分析指数膨胀的L1表达水平和表达的L1位点的数量。应用SCIFER分析小鼠和人睾丸产生的scRNA-Seq数据集，发现小鼠圆形精子细胞和人精原细胞、精母细胞和圆形精子细胞表达最高水平的L1 mRNA。我们的分析还确定，与小鼠相似，来自无关个体的人类睾丸共享多达80%的表达的L1基因座。此外，SCIFER确定单个小鼠细胞共表达不同的L1亚家族和不同的转座因子家族，实验验证了它们在同一细胞中的共存。SCIFER检测单个细胞中单个L1基因座的mRNA表达。它与使用传统测序方法制备的scRNA-Seq数据集兼容。使用人类癌细胞系进行验证，小鼠和人类睾丸的SCIFER分析确定了支持这些物种中L1表达的关键细胞类型。这将进一步加深我们对小鼠和人类内源性L1 mRNA表达模式的差异和相似性的理解。在线版本包含补充材料，可通过10.1186/s13100-022-00276-0获得。
Endogenous expression of L1 mRNA is the first step in an L1-initiated mutagenesis event. However, the contribution of individual cell types to patterns of organ-specific L1 mRNA expression remains poorly understood, especially at single-locus resolution. We introduce a method to quantify expression of mobile elements at the single-locus resolution in scRNA-Seq datasets called Single Cell Implementation to Find Expressed Retrotransposons (SCIFER). SCIFER aligns scRNA-Seq reads uniquely to the genome and extracts alignments from single cells by cell-specific barcodes. In contrast to the alignment performed using default parameters, this alignment strategy increases accuracy of L1 locus identification by retaining only reads that are uniquely mapped to individual L1 loci. L1 loci expressed in single cells are unambiguously identified using a list of L1 loci manually validated to be expressed in bulk RNA-Seq datasets generated from the same cell line or organ. Validation of SCIFER using MCF7 cells determined technical parameters needed for optimal detection of L1 expression in single cells. We show that unsupervised analysis of L1 expression in single cells exponentially inflates both the levels of L1 expression and the number of expressed L1 loci. Application of SCIFER to analysis of scRNA-Seq datasets generated from mouse and human testes identified that mouse Round Spermatids and human Spermatogonia, Spermatocytes, and Round Spermatids express the highest levels of L1 mRNA. Our analysis also determined that similar to mice, human testes from unrelated individuals share as much as 80% of expressed L1 loci. Additionally, SCIFER determined that individual mouse cells co-express different L1 sub-families and different families of transposable elements, experimentally validating their co-existence in the same cell. SCIFER detects mRNA expression of individual L1 loci in single cells. It is compatible with scRNA-Seq datasets prepared using traditional sequencing methods. Validated using a human cancer cell line, SCIFER analysis of mouse and human testes identified key cell types supporting L1 expression in these species. This will further our understanding of differences and similarities in endogenous L1 mRNA expression patterns in mice and humans. The online version contains supplementary material available at 10.1186/s13100-022-00276-0.
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影响因子：
14.9
作者：
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