title: Hybrid sequencing and map finding (HySeMaFi): optional strategies for extensively deciphering gene splicing and expression in organisms without reference genome.
abstract: Using second-generation sequencing (SGS) RNA-Seq strategies, extensive alterative splicing prediction is impractical and high variability of isoforms expression quantification is inevitable in organisms without true reference dataset. we report the development of a novel analysis method, termed hybrid sequencing and map finding (HySeMaFi) which combines the specific strengths of third-generation sequencing (TGS) (PacBio SMRT sequencing) and SGS (Illumina Hi-Seq/MiSeq sequencing) to effectively decipher gene splicing and to reliably estimate the isoforms abundance. Error-corrected long reads from TGS are capable of capturing full length transcripts or as large partial transcript fragments. Both true and false isoforms, from a particular gene, as well as that containing all possible exons, could be generated by employing different assembly methods in SGS. We first develop an effective method which can establish the mapping relationship between the error-corrected long reads and the longest assembled contig in every corresponding gene. According to the mapping data, the true splicing pattern of the genes was reliably detected, and quantification of the isoforms was also effectively determined. HySeMaFi is also the optimal strategy by which to decipher the full exon expression of a specific gene when the longest mapped contigs were chosen as the reference set.
authors.affiliation: Key laboratory of Horticultural Plant Biology, Ministry of Education, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, P. R. China.
authors.forename: Guogui
authors.initials: G
authors.lastname: Ning
authors.affiliation: Medical Research Institute, School of Medicine, Wuhan University, Wuhan, P. R. China.
authors.forename: Xu
authors.initials: X
authors.lastname: Cheng
authors.affiliation: Key laboratory of Horticultural Plant Biology, Ministry of Education, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, P. R. China.
authors.forename: Ping
authors.initials: P
authors.lastname: Luo
authors.affiliation: Nextomics Biosciences Co., Ltd., Wuhan, Hubei, China.
authors.forename: Fan
authors.initials: F
authors.lastname: Liang
authors.affiliation: Key laboratory of Horticultural Plant Biology, Ministry of Education, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, P. R. China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Key laboratory of Horticultural Plant Biology, Ministry of Education, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, P. R. China.
authors.forename: Guoliang
authors.initials: G
authors.lastname: Yu
authors.affiliation: Key laboratory of Horticultural Plant Biology, Ministry of Education, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, P. R. China.
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.affiliation: Nextomics Biosciences Co., Ltd., Wuhan, Hubei, China.
authors.forename: Depeng
authors.initials: D
authors.lastname: Wang
authors.affiliation: Key laboratory of Horticultural Plant Biology, Ministry of Education, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, P. R. China.
authors.forename: Manzhu
authors.initials: M
authors.lastname: Bao
mesh_terms: D017398:Alternative Splicing; D019295:Computational Biology / Q000379:methods; D020451:Contig Mapping / Q000379:methods*; D005091:Exons / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D018506:Gene Expression Regulation, Plant; D023281:Genomics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D032306:Petunia / Q000235:genetics; D059368:RNA Isoforms / Q000235:genetics; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D059368:RNA Isoforms