RNA-seq is a well-established method for studying the transcriptome. Popular methods for library preparation in RNA-seq such as Illumina TruSeq® RNA v2 kit use a poly-A pulldown strategy. Such methods can cause loss of coverage at the 5′ end of genes, impacting the ability to detect fusions when used on degraded samples. The goal of this study was to quantify the effects RNA degradation has on fusion detection when using poly-A selected mRNA and to identify the variables involved in this process.
Using both artificially and naturally degraded samples, Mayo Clinic researchers found that there is a reduced ability to detect fusions as the distance of the breakpoint from the 3′ end of the gene increases. The median transcript coverage decreases exponentially as a function of the distance from the 3′ end and there is a linear relationship between the coverage decay rate and the RNA integrity number (RIN). Based on these findings the researchers developed plots that show the probability of detecting a gene fusion (“sensitivity”) as a function of the distance of the fusion breakpoint from the 3′ end.
Estimation of fusion sensitivity as a function of distance of breakpoint from the 3′ end
a Diagram showing the strategy used to estimate the probability of detecting a fusion (i.e. sensitivity) at different distances from the 3′ end by enumerating the proportion of genes having a coverage ≥10x. A indicates that there are more than 10 reads at that particular position for that gene while a X indicates that there are not. b Sensitivity as a function of the distance from the 3′ end for the UHR sample at varying levels of degradation. Loess trend is shown for each sample and 95 % confidence intervals are shown in gray. c Estimated sensitivity (log scale) for five different fusions present in UHR that occur at different distances from the 3′ end at different RIN values. If the fusion was detected at the particular degradation level it is shown as a square and if it is not detected it is shown as a circle. Linear trend line is shown for each fusion and 95 % confidence intervals are shown in gray