title: Optimizing and benchmarking de novo transcriptome sequencing: from library preparation to assembly evaluation.
abstract: BACKGROUND
  RNA-seq enables gene expression profiling in selected spatiotemporal windows and yields massive sequence information with relatively low cost and time investment, even for non-model species. However, there remains a large room for optimizing its workflow, in order to take full advantage of continuously developing sequencing capacity.
  
  
  METHODS
  Transcriptome sequencing for three embryonic stages of Madagascar ground gecko (Paroedura picta) was performed with the Illumina platform. The output reads were assembled de novo for reconstructing transcript sequences. In order to evaluate the completeness of transcriptome assemblies, we prepared a reference gene set consisting of vertebrate one-to-one orthologs.
  
  
  RESULTS
  To take advantage of increased read length of >150 nt, we demonstrated shortened RNA fragmentation time, which resulted in a dramatic shift of insert size distribution. To evaluate products of multiple de novo assembly runs incorporating reads with different RNA sources, read lengths, and insert sizes, we introduce a new reference gene set, core vertebrate genes (CVG), consisting of 233 genes that are shared as one-to-one orthologs by all vertebrate genomes examined (29 species)., The completeness assessment performed by the computational pipelines CEGMA and BUSCO referring to CVG, demonstrated higher accuracy and resolution than with the gene set previously established for this purpose. As a result of the assessment with CVG, we have derived the most comprehensive transcript sequence set of the Madagascar ground gecko by means of assembling individual libraries followed by clustering the assembled sequences based on their overall similarities.
  
  
  CONCLUSIONS
  Our results provide several insights into optimizing de novo RNA-seq workflow, including the coordination between library insert size and read length, which manifested in improved connectivity of assemblies. The approach and assembly assessment with CVG demonstrated here would be applicable to transcriptome analysis of other species as well as whole genome analyses.
authors.affiliation: Phyloinformatics Unit, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima-minami, Chuo-ku, Kobe, Hyogo, 650-0047, Japan. yuichiro.hara@riken.jp.
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Hara
authors.affiliation: Phyloinformatics Unit, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima-minami, Chuo-ku, Kobe, Hyogo, 650-0047, Japan. kaori.tatsumi@riken.jp.
authors.forename: Kaori
authors.initials: K
authors.lastname: Tatsumi
authors.affiliation: Laboratory for Vertebrate Body Plan, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minami, Chuo-ku, Kobe, Hyogo, 650-0047, Japan. myoshida@cdb.riken.jp.
authors.forename: Michio
authors.initials: M
authors.lastname: Yoshida
authors.affiliation: Laboratory for Vertebrate Body Plan, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minami, Chuo-ku, Kobe, Hyogo, 650-0047, Japan. kajikawa@cdb.riken.jp.
authors.forename: Eriko
authors.initials: E
authors.lastname: Kajikawa
authors.affiliation: Animal Resource Development Unit, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima-minami, Chuo-ku, Kobe, Hyogo, 650-0047, Japan. hkiyo@cdb.riken.jp.|Genetic Engineering Team, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima-minami, Chuo-ku, Kobe, Hyogo, 650-0047, Japan. hkiyo@cdb.riken.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kiyonari
authors.affiliation: Phyloinformatics Unit, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima-minami, Chuo-ku, Kobe, Hyogo, 650-0047, Japan. shigehiro.kuraku@riken.jp.
authors.forename: Shigehiro
authors.initials: S
authors.lastname: Kuraku
mesh_terms: D000818:Animals; D019985:Benchmarking; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D009928:Organ Specificity; D012015:Reference Standards; D012104:Reptiles / Q000196:embryology / Q000235:genetics; D017423:Sequence Analysis, RNA