Computational protocol: Transcriptome Analysis Comparison of Lipid Biosynthesis in the Leaves and Developing Seeds of Brassica napus

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Protocol publication

[…] The raw reads were first filtered by discarding the reads with adaptor contamination, low-quality sequences (reads with ambiguous ‘N’ bases), and reads with more than 10% Q < 20 bases. Then the clean reads were assembled into contigs using the Trinity program [], which efficiently reconstructed full-length transcripts across a broad range of expression levels and sequencing depths. Subsequently, the contigs were linked into transcripts according to the paired-end information of the sequences, and the transcripts were clustered based on nucleotide sequence identity. The longest transcripts in the cluster units were regarded as unigenes in order to eliminate redundant sequences, and then the unigenes were combined to produce the final assembly used for annotation. The unigenes information was deposited in the Sequence Read Archive (SRA) database in NCBI (Accession number, SRR1916242).To understand their functions, the unigenes were annotated using BLASTx alignment, with an E-value cut-off of 10–5, against the NCBI non-redundant (NR) database, and the UniProt/Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG), Cluster of Orthologous Groups of proteins (COG) and Gene Ontology (GO) databases.The RPKM (Reads Per Kilobase per Million mapped reads) method was used to calculate unigenes’ expression []. The RPKM method is able to reflect the molar concentration of a transcript by normalizing for RNA length and for the total read number. We compared the unigenes expressions using their RPKM values. […]

Pipeline specifications

Software tools Trinity, BLASTX
Databases UniProt KEGG
Application RNA-seq analysis
Organisms Brassica napus
Chemicals Fatty Acids, Triglycerides, alpha-Linolenic Acid, Oleic Acid, Palmitic Acid, Linoleic Acid