I started learning how to use Tophat tool. I used it to check RNA sequencing and I calculated 64.91% in the percentage of uniquely aligned reads. I read in article that in RNA sequencing we should expect to get 80% or higher of uniquely aligened reads to get a good read but I didnt understand the reason why.
I conducted many RNA-seq analyis, however, the ratio of uniquely aligned reads was 60-70% in most samples. In other case, the mapping ratio (including unique mapping and multiple mapping) was over 90%. In my opinion, the unique mapping ratio was affected by many factors, including sequencing, tools, and species ect.. In plant, there are many repeat sequence in their genome, so, the unique mapping aslo is relatively low compared with animal.
Uniquely mapped reads are the only way to know for sure where a specific read came from. When you have ambiguously mapped reads, it's virtually impossible to know that. There's no specific number that works for all organisms, you should evaluate case by case since technical and biological factors will influence the % of ambiguously-mapped reads. There are several computational methods to handle these multi-mapped reads. I believe this paper will better answer all your questions on this topic: Article Handling multi-mapped reads in RNA-seq
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