[轉錄本豐度和tximport管道

在我們演示如何對齊然后計數(shù)RNA seq片段之前，我們提到一種更新更快的替代管道是使用轉錄豐度量化方法，如Sailfish8、Salmon9、kallisto10或RSEM11，在不對齊讀取的情況下估計豐度，然后是tximport軟件包，用于組裝計數(shù)矩陣和偏移矩陣，用于Bioconductor差異基因表達軟件包。我們已將其作為該工作流程修訂過程的一部分添加，因此以下材料涵蓋了通過對齊和讀取/碎片計數(shù)生成計數(shù)矩陣。將轉錄物豐度量詞與tximport結合使用以產生基因水平計數(shù)矩陣和標準化偏移量的優(yōu)點是：該方法校正了樣本間基因長度的任何潛在變化（例如，來自差異異構體的使用）12；與基于對齊的方法相比，其中一些方法速度更快，所需的內存和磁盤使用量更少；而且可以避免丟棄那些可以與多個具有同源序列的基因對齊的片段13。請注意，成績單豐度量詞跳過存儲讀取比對的大型文件的生成，而生成存儲每個成績單的估計豐度、計數(shù)和有效長度的較小文件。有關更多詳細信息，請參閱描述tximport方法的手稿14和tximport包漸暈圖以了解軟件詳細信息。在使用成績單量詞和tximport之后，返回此工作流的入口點將是下面數(shù)據(jù)對象的部分。

Transcript abundances and the tximport pipeline

Before we demonstrate how to align and then count RNA-seq fragments, we mention that a newer and faster alternative pipeline is to use transcript abundance quantification methods such as Sailfish8, Salmon9, kallisto10 or RSEM11 to estimate abundances without aligning reads, followed by the tximport package for assembling count and offset matrices for use with Bioconductor differential gene expression packages. We have added this as part of the revision process for this workflow, therefore the following material covers generation of count matrices through alignment and read/fragment counting. The advantages of using the transcript abundance quantifiers in conjunction with tximport to produce gene-level count matrices and normalizing offsets are: this approach corrects for any potential changes in gene length across samples (e.g., from differential isoform usage)12; some of these methods are substantially faster and require less memory and disk usage compared to alignment-based methods; and it is possible to avoid discarding those fragments that can align to multiple genes with homologous sequence13. Note that transcript abundance quantifiers skip the generation of large files which store read alignments, instead producing smaller files which store estimated abundances, counts, and effective lengths per transcript. For more details, see the manuscript describing the tximport approach14 and the tximport package vignette for software details. The entry point back into this workflow after using a transcript quantifier and tximport would be the section on the data object below.