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Protocol publication

[…] FastQ files from the sequencing run were de-multiplexed using CASAVA 1.8.2 software (Illumina). Read quality as determined by ‘phred’ scores was assessed using FastQC v0.11.4 () on the LIMS High Performance Computing cluster (La Trobe University, Bundoora, Australia). High quality reads for all treatments, including the rhizome reads, were de novo assembled using Trinity version r20140717 () with stranded data and a minimum k-mer coverage of one to produce a reference transcriptome. Reads from individual samples were then mapped back to the reference using Bowtie (). The number of reads per gene model (hereafter referred to as a gene) was determined using RSEM () before annotation performed in Trinotate. BLAST searches included Blastp and Blastx against the SwissProt database and Blastx nr and Blastn nt against the GenBank database.Analysis of differential gene expression among treatments for culm samples was undertaken using edgeR v3.10.5 () as implemented in Degust v0.2 with the False Discovery Rate (FDR) cut-off initially set to 1.0 and then to 0.05 for subsequent analyses. Using the latter FDR value, differences in expression levels of genes common to different treatments were assessed for samples grouped by source site salinity class (Table ) and by irrigation treatment. Throughout the Results section, ‘L’ refers to plants sourced from low salinity sites and ‘H’ refers to plants sourced from high salinity sites as per Table . Salinity treatments were coded as ‘0’ for freshwater and ‘16’ for 16 g L-1 TDS. Gene expression levels from plants sourced from low salinity sites and irrigated with fresh water (L-0) were used as a baseline for comparison. Output from Degust was used to construct four-way Venn diagrams using Venny v.2.0.2 () to highlight differentially expressed genes common (or unique) to comparisons between specific pair-wise interactions of different treatments. The comparisons presented below highlight informative differences between plants sourced from ‘L’ and ‘H’ salinity sites and their response to irrigation with ‘0’ and ‘16’ treatments. […]

Pipeline specifications

Software tools BaseSpace, FastQC, Trinity, Bowtie, RSEM, Trinotate, BLASTP, BLASTX, BLASTN, edgeR, VENNY
Organisms Phragmites australis, Bhavania australis