Identifying differential isoform abundance with RATs: a universal tool and a warning
Identifying differential isoform abundance with RATs: a universal tool and a warning
复制标题
DOI：
10.1101/132761
复制
发表时间：
2017-05
期刊：
影响因子：
--
通讯作者：
Kimon Froussios;Kira Mourão;G. Simpson;G. Barton;N. Schurch
中科院分区：
文献类型：
--
作者：
Kimon Froussios;Kira Mourão;G. Simpson;G. Barton;N. Schurch
基因和转录本表达变化的生物学重要性是公认的，并反映了各种各样的工具，可用于验证这些变化。通过差异转录本使用（DTU）的调节正在成为一个重要的现象。存在几种用于从读段比对或组装数据检测DTU的工具，但是用于从无干扰定量检测DTU的选项是有限的。结果我们提出了一个R软件包，名为RAT-（相对Abstraction of Transcripts）-直接从转录本丰度估计中识别DTU转录组范围。RAT对于量化方法是不可知的，并且利用自举量化（如果可用的话）来通知检测到的DTU事件的重要性。RAT将DTU结果置于背景中，并在所有复制级别显示出良好的错误发现性能（中位数FDR ≤0.05）。我们将RAT应用于与特发性肺纤维化相关的人类RNA-seq数据集，其中三个DTU事件通过qRT-PCR验证。RAT发现，基于Ensembl v60注释，所有三种基因的亚型比例均表现出统计学显著性变化，但两种基因的DTU在自举定量中未可靠重现。RAT还确定了500个新的DTU事件，这些事件富含与刺激反应调节、免疫系统过程调节和共生/寄生有关的11个GO术语。用Ensembl v87注释重复该分析显示，三个验证的DTU基因中的两个的同种型丰度谱发生了根本性变化。RAT确定了414个新的DTU事件，这些事件富集了五个GO术语，其中没有一个与先前确定的事件相同。在两项分析中，只有141例DTU事件是常见的，而在原始研究报告的248例事件中，只有8例是常见的。此外，原始qRT-PCR探针不再与其原始转录物唯一匹配，从而对这些数据的解释提出了质疑。我们建议平行全长亚型测序，注释预过滤和测序的转录本捕获的qRT-PCR引物作为可能的方法，以提高验证RNA-seq结果在未来的实验。可用性该软件包可通过Github网站https://github.com/bartongroup/Rats获得。
Motivation The biological importance of changes in gene and transcript expression is well recognised and is reflected by the wide variety of tools available to characterise these changes. Regulation via Differential Transcript Usage (DTU) is emerging as an important phenomenon. Several tools exist for the detection of DTU from read alignment or assembly data, but options for detection of DTU from alignment-free quantifications are limited. Results We present an R package named RATs – (Relative Abundance of Transcripts) – that identifies DTU transcriptome-wide directly from transcript abundance estimations. RATs is agnostic to quantification methods and exploits bootstrapped quantifications, if available, to inform the significance of detected DTU events. RATs contextualises the DTU results and shows good False Discovery performance (median FDR ≤0.05) at all replication levels. We applied RATs to a human RNA-seq dataset associated with idiopathic pulmonary fibrosis with three DTU events validated by qRT-PCR. RATs found all three genes exhibited statistically significant changes in isoform proportions based on Ensembl v60 annotations, but the DTU for two were not reliably reproduced across bootstrapped quantifications. RATs also identified 500 novel DTU events that are enriched for eleven GO terms related to regulation of the response to stimulus, regulation of immune system processes, and symbiosis/parasitism. Repeating this analysis with the Ensembl v87 annotation showed the isoform abundance profiles of two of the three validated DTU genes changed radically. RATs identified 414 novel DTU events that are enriched for five GO terms, none of which are in common with those previously identified. Only 141 of the DTU evens are common between the two analyses, and only 8 are among the 248 reported by the original study. Furthermore, the original qRT-PCR probes no longer match uniquely to their original transcripts, calling into question the interpretation of these data. We suggest parallel full-length isoform sequencing, annotation pre-filtering and sequencing of the transcripts captured by qRT-PCR primers as possible ways to improve the validation of RNA-seq results in future experiments. Availability The package is available through Github at https://github.com/bartongroup/Rats.