title: Impact of RNA degradation on fusion detection by RNA-seq.
abstract: 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.
authors.affiliation: Department of Health Science Research, Mayo Clinic, Rochester, MN, 55905, USA. davila.jaime@mayo.edu.
authors.forename: Jaime I
authors.initials: JI
authors.lastname: Davila
authors.affiliation: Department of Health Science Research, Mayo Clinic, Rochester, MN, 55905, USA.
authors.forename: Numrah M
authors.initials: NM
authors.lastname: Fadra
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, 55905, USA.
authors.forename: Xiaoke
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, 55905, USA.
authors.forename: Amber M
authors.initials: AM
authors.lastname: McDonald
authors.affiliation: Department of Health Science Research, Mayo Clinic, Rochester, MN, 55905, USA.
authors.forename: Asha A
authors.initials: AA
authors.lastname: Nair
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, 55905, USA.
authors.forename: Barbara R
authors.initials: BR
authors.lastname: Crusan
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, 55905, USA.
authors.forename: Xianglin
authors.initials: X
authors.lastname: Wu
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, 55905, USA.
authors.forename: Joseph H
authors.initials: JH
authors.lastname: Blommel
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, 55905, USA.|Genome Analysis Core, Medical Genome Facility, Center for Individualized Medicine, Mayo Clinic, Rochester, MN, 55905, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Jen
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, 55905, USA.
authors.forename: Kandelaria M
authors.initials: KM
authors.lastname: Rumilla
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, 55905, USA.
authors.forename: Robert B
authors.initials: RB
authors.lastname: Jenkins
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, 55905, USA.
authors.forename: Umut
authors.initials: U
authors.lastname: Aypar
authors.affiliation: Department of Health Science Research, Mayo Clinic, Rochester, MN, 55905, USA.|Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, 55905, USA.
authors.forename: Eric W
authors.initials: EW
authors.lastname: Klee
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, 55905, USA.
authors.forename: Benjamin R
authors.initials: BR
authors.lastname: Kipp
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, 55905, USA.
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Halling
mesh_terms: D045744:Cell Line, Tumor; D056905:Chromosome Breakpoints; D016044:Fusion Proteins, bcr-abl / Q000235:genetics; D015723:Gene Library; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics; D012313:RNA / Q000235:genetics* / Q000378:metabolism; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics; D011995:Recombination, Genetic; D017423:Sequence Analysis, RNA
chemical_list: D012333:RNA, Messenger; D012313:RNA; D016044:Fusion Proteins, bcr-abl
keywords: Fusion detection; Poly-A pulldown; RNA degradation; RNA-seq