title: Comparison of different sequencing and assembly strategies for a repeat-rich fungal genome, Ophiocordyceps sinensis.
abstract: Ophiocordyceps sinensis is one of the most expensive medicinal fungi world-wide, and has been used as a traditional Chinese medicine for centuries. In a recent report, the genome of this fungus was found to be expanded by extensive repetitive elements after assembly of Roche 454 (223Mb) and Illumina HiSeq (10.6Gb) sequencing data, producing a genome of 87.7Mb with an N50 scaffold length of 12kb and 6972 predicted genes. To test whether the assembly could be improved by deeper sequencing and to assess the amount of data needed for optimal assembly, genomic sequencing was run several times on genomic DNA extractions of a single ascospore isolate (strain 1229) on an Illumina HiSeq platform (25Gb total data). Assemblies were produced using different data types (raw vs. trimmed) and data amounts, and using three freely available assembly programs (ABySS, SOAP and Velvet). In nearly all cases, trimming the data for low quality base calls did not provide assemblies with higher N50 values compared to the non-trimmed data, and increasing the amount of input data (i.e. sequence reads) did not always lead to higher N50 values. Depending on the assembly program and data type, the maximal N50 was reached with between 50% to 90% of the total read data, equivalent to 100× to 200× coverage. The draft genome assembly was improved over the previously published version resulting in a 114Mb assembly, scaffold N50 of 70kb and 9610 predicted genes. Among the predicted genes, 9213 were validated by RNA-Seq analysis in this study, of which 8896 were found to be singletons. Evidence from genome and transcriptome analyses indicated that species assemblies could be improved with defined input material (e.g. haploid mono-ascospore isolate) without the requirement of multiple sequencing technologies, multiple library sizes or data trimming for low quality base calls, and with genome coverages between 100× and 200×.
authors.affiliation: State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, NO. 1 Beichen West Road, Chaoyang District, Beijing 100101, China; Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agricultural and Forestry University, No.15 Shangxiadian Road, Cangshan District, Fuzhou 350002, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Li
authors.affiliation: School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Tom
authors.initials: T
authors.lastname: Hsiang
authors.affiliation: State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, NO. 1 Beichen West Road, Chaoyang District, Beijing 100101, China; University of Chinese Academy of Sciences, 19 A Yuquan Rd, Shijingshan District, Beijing 100049, China.
authors.forename: Rui-Heng
authors.initials: RH
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, NO. 1 Beichen West Road, Chaoyang District, Beijing 100101, China; University of Chinese Academy of Sciences, 19 A Yuquan Rd, Shijingshan District, Beijing 100049, China.
authors.forename: Xiao-Di
authors.initials: XD
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, NO. 1 Beichen West Road, Chaoyang District, Beijing 100101, China; University of Chinese Academy of Sciences, 19 A Yuquan Rd, Shijingshan District, Beijing 100049, China.
authors.forename: Ke
authors.initials: K
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, NO. 1 Beichen West Road, Chaoyang District, Beijing 100101, China.
authors.forename: Wen-Jing
authors.initials: WJ
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, NO. 1 Beichen West Road, Chaoyang District, Beijing 100101, China.
authors.forename: Xiao-Liang
authors.initials: XL
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, NO. 1 Beichen West Road, Chaoyang District, Beijing 100101, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Jiao
authors.affiliation: State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, NO. 1 Beichen West Road, Chaoyang District, Beijing 100101, China. Electronic address: yaoyj@im.ac.cn.
authors.forename: Yi-Jian
authors.initials: YJ
authors.lastname: Yao
mesh_terms: D004271:DNA, Fungal / Q000235:genetics; D020869:Gene Expression Profiling; D016681:Genome, Fungal; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006999:Hypocreales / Q000235:genetics*; D012331:RNA, Fungal / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004271:DNA, Fungal; D012331:RNA, Fungal
keywords: Assembly; Coverage; Genome; RNA-Seq; Sequencing; Trimming