Rubus GDR RefTrans V1

Overview
Analysis NameRubus GDR RefTrans V1
Methodreftrans (1.0)
Sourcerubus_GDR_reftransV1
Date performed2016-07-14

Materials & Methods

GDR Rubus RefTrans combines published RNA-Seq and EST data sets to create a reference transcriptome (RefTrans) for rubus and provides putative gene function identified by homology to known proteins.   

In  Rubus_RefTrans_V1, 265 million Illumina paired-end reads from publicly available peer-reviewed rubus RNA-Seq data sets (Hyun et al. 2014,  Hyun et al. 2014Garcia-Seco et al. 2015),  and 3,184 ESTs, were downloaded from the NCBI Short Read Archive database (SRR975478, SRX347804), the EBI database (PRJEB6680)  and the NCBI dbEST database, respectively. The RNA-Seq sequences were subjected to quality control using the NGS QC Toolkit (V2.3.3, default parameters, Patel and Jain, 2012) and custom Perl scripts. The remaining 264 million RNA-Seq reads were assembled de novo with Trinity (v2.0.6, Grabherr et al, 2011) using default assembly parameters and a minimum coding length of 200 bases. Quality control of the ESTs included vector sequence screening (UniVec_Core,ftp://ftp.ncbi.nih.gov/pub/UniVec/) using cross_match (Gordon et al, 1998), removal of tRNA/rRNA/snRNA sequences identified using tblastx (Altschul et al, 1990), and Poly-A tail trimmimg.  The filtered ESTs were assembled using the CAP3 program (P -90, Huan and Madan, 1999). Bowtie (v 2-2.2.3) (Langmead et al, 2009) was applied to multi-map the RNA-Seq reads and ESTs back to the assembled contigs and singlets. The contigs and singlets were hierarchically clustered into genes using Corset (v1.0.4) (Davidson and Oshlack, 2014) with default parameters. The longest isoform was selected to represent each Corset cluster, creating a RefTrans V1 for rubus of  37,326 sequences. The RefTrans were functionally characterized by pairwise comparison using the BLASTX algorithm against the Swiss-Prot (UniProtKB/Swiss-Prot Release 2015_10) and TrEMBL (UniProtKB/TrEMBL Release 2015_10) (Boeckmann et al, 2003) protein databases.  Information on the top 25 matches with an expect (E) value of ≤ 1E-06 were recorded and stored in the database. The transcriptome and annotation (GO Terms, match description, InterPro domains) are available for searching and downloading.   

 

References:

  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. (1990) Basic local alignment search tool. J Mol Biol. 215(3):403-10.

  2. Boeckmann B., Bairoch A., Apweiler R., Blatter M.-C., Estreicher A., Gasteiger E., Martin M.J., Michoud K., O'Donovan C., Phan I., Pilbout S., and Sneider M. (2003) The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003. Nucleic acids. 31:365-370.

  3. Davidson, N. M. and Oshlack, A. (2014). Corset: enabling differential gene expression analysis forde novo assembled transcriptomes. Genome Bio. 15(7):410 

  4. Gordon D, Abajian C, Green P. (1998) Consed: a graphical tool for sequence finishing.  Genome Res. 1998 Mar;8(3):195-202.

  5. Grabherr MG, Haas BJ, Yassour M, et al. (2011) Trinity: reconstructing a full-length transcriptome without a genome from RNA-Seq data. Nat. Biotechnol. 29(7):644-652.

  6. Huan, X. and Madan, A. (1999). CAP3: A DNA sequence assembly program. Genome Research, 9, 868-877.

  7. Langmead, B., Cole Trapnell, C., Pop, M. and Salzberg, S.L. (2009) Bowtie Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Bio. 10:R25 doi:10.1186/gb-2009-10-3-r25

  8. Patel RK, Jain M (2012). NGS QC Toolkit: A toolkit for quality control of next generation sequencing data. PLoS ONE, 7(2): e30619
Properties
Additional information about this analysis:
Property NameValue
Analysis Typereftrans