stranded 和 non-stranded RNA-seq?的表達譜分析和基因overlap研究的比較

原文鏈接：Comparison of stranded and non-stranded RNA-seq transcriptome profiling and investigation of gene overlap

背景

non-stranded RNA-seq的建庫存在的問題：不能確定每個轉錄本(transcript)是來自哪條鏈，沒有鏈特異信息，則難以準確量化基因的表達水平，尤其是在genomic loci有重疊，但是轉錄方向是相反的這些基因。

strand-specific or stranded RNA-seq的建庫方式能夠保留鏈的信息。

實驗結果

材料：whole blood RNA samples?

處理：stranded 和 non-stranded RNA-seq?

結果：stranded 和 non-stranded RNA-seq?的基因表達分析表明，stranded RNA-seq 能識別更多的差異表達基因。這主要是因為能夠更準確的估計在genomic loci有重疊，但是轉錄方向是相反的基因的表達水平。

建議：使用Stranded RNA-seq方式建庫。

Non-stranded 和 stranded RNA-seq 建庫步驟的比較

區別：

（1）cDNA?合成中，第二條鏈合成是否使用dUTPs代替dTTPs；

（2）文庫建成后，是否降解第二條鏈。

例1：

The mapping profiles for?IL24?in Replicate PFE1. In non-stranded RNA-seq, all reads mapped to?IL24?are counted regardless if they are in the forward or reverse strands. However, in stranded RNA-seq, nearly all reads are mapped to the “+” strand and thus not counted because these reads are not reverse complementary to IL24 in the “+” strand. However, the coverage pattern of sequence reads does not support the sequence reads mapped to the?IL24?genomic region that truly originate from this gene. All genes, transcripts, and sequence reads are colored in blue if they are in the “+” strand and colored in green if in the “?“ strand

例2：

The mapping profiles for?ICAM4?(intercellular adhesion molecule 4) in Replicate PFE1. The gene?ICAM4?is on the “+” strand, and 100?% contained within?CTD-2369P2.8?in the “?“ strand. In non-stranded RNA-seq, the ambiguous reads in overlapping regions are excluded from counting, which explains why there is no expression for?ICAM4. However, the ambiguous reads can be perfectly resolved in stranded RNA-seq. By considering the read direction, all reads can be counted to?ICAM4?because they are reverse complementary to?ICAM4, but not?CTD-2369P2.8

轉載于:https://www.cnblogs.com/adawong/articles/8033860.html

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