Impact of RNA degradation on fusion detection by RNA-seq

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 whe...

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Bibliographic Details
Published in:BMC genomics 2016-10, Vol.17 (1), p.814-814, Article 814
Main Authors: Davila, Jaime I, Fadra, Numrah M, Wang, Xiaoke, McDonald, Amber M, Nair, Asha A, Crusan, Barbara R, Wu, Xianglin, Blommel, Joseph H, Jen, Jin, Rumilla, Kandelaria M, Jenkins, Robert B, Aypar, Umut, Klee, Eric W, Kipp, Benjamin R, Halling, Kevin C
Format: Article
Language:English
Subjects:
Cell Line, Tumor
Chromosome Breakpoints
Fusion Proteins, bcr-abl - genetics
Gene Library
Genes
Genetic transcription
Humans
Leukemia, Myelogenous, Chronic, BCR-ABL Positive - genetics
Messenger RNA
Recombination, Genetic
RNA - genetics
RNA - metabolism
RNA sequencing
RNA Stability
RNA, Messenger - genetics
Sequence Analysis, RNA
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Summary: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, we 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 we 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. This study developed a strategy to assess the impact that RNA degradation has on the ability to detect gene fusions by RNA-seq.
ISSN:1471-2164
1471-2164
DOI:10.1186/s12864-016-3161-9