Optimizing and benchmarking de novo transcriptome sequencing: from library preparation to assembly evaluation

BMC Genomics

Research Article

Hiroshi Kiyonari¹  Michio Yoshida²  Eriko Kajikawa²  Shigehiro Kuraku³  Kaori Tatsumi³  Yuichiro Hara³ 

[1] Animal Resource Development Unit, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima-minami, Chuo-ku, 650-0047, Kobe, Hyogo, Japan;Genetic Engineering Team, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima-minami, Chuo-ku, 650-0047, Kobe, Hyogo, Japan;Laboratory for Vertebrate Body Plan, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minami, Chuo-ku, 650-0047, Kobe, Hyogo, Japan;Phyloinformatics Unit, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima-minami, Chuo-ku, 650-0047, Kobe, Hyogo, Japan;

关键词: RNA-seq;    Transcriptome sequencing;    de novo;    Completeness assessment;    Library insert length;    CVG (core vertebrate genes);    Madagascar ground gecko;   

DOI  :  10.1186/s12864-015-2007-1

 received in 2015-04-06, accepted in 2015-10-03,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundRNA-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.MethodTranscriptome 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.ResultTo 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.ConclusionOur 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.

【 授权许可】

CC BY   
© Hara et al. 2015