Computational protocol: Small RNA Profile in Moso Bamboo Root and Leaf Obtained by High Definition Adapters

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Protocol publication

[…] We used version 1.0 of the bamboo genome and the annotations available on the ICBR-CAFNET website (http://202.127.18.221/bamboo/down.php). We downloaded the bamboo chloroplast genome, accession NC_015817 from (http://chloroplast.ocean.washington.edu) and used the publicly available annotations . First, the fastq files from NGS were converted to fasta files and sequence reads with no Ns were kept for further analysis. Next, the first 8 nt of the 3′ adapters were identified and removed and then four nucleotides on the 5′ and 3′ ends of the reads were trimmed (which corresponded to the NNNN tags on the HD adapters; see: Sorefan et al. 2012) using the UEA sRNA Workbench . The sequence reads were mapped to the bamboo genome and the bamboo chloroplast genome with 0 mis-matches, in non-redundant format, using PatMaN . The abundance of sequenced reads was normalized to the total number of reads in the sample, as indicated in . The differentially expressed reads were identified using offset fold change as described in , using an offset of 20. The miRNAs were identified using the miRCat and miRProf tools within the sRNA Workbench and custom made Perl scripts (Strawberry Perl v 5.18.2.1). The miRNA targets were predicted using the target prediction tool within the UEA sRNA Workbench . The rules used for target prediction are based on those suggested by Allen et al. and Schwab et al. , . Specifically, no more than four mismatches between miRNA and target (G-U bases count as 0.5 mismatches) are allowed, no more than two adjacent mismatches in the miRNA/target duplex are permitted, no adjacent mismatches in positions 2–12 of the miRNA/target duplex (5′ of miRNA) and no more than 2.5 mismatches in positions 1–12 are accepted, no mismatches in positions 10–11 of miRNA/target duplex are permitted. In addition, the minimum free energy (MFE) of the miRNA/target duplex should be > = 74% of the MFE of the miRNA bound to its perfect complement. […]

Pipeline specifications

Software tools PatMaN, miRCat, miRProf
Application Nucleotide sequence alignment
Organisms Phyllostachys edulis