RNA-Seq Analysis of Fom-5190a Genome

RNA integrity was confirmed using the Agilent 2100 Bioanalyser Plant Nano system (Agilent Biotechnologies). Stranded Illumina TruSeq libraries were generated from 1 μg of total RNA and sequenced (100 bp paired end reads) on an Illumina HiSeq platform by the Australian Genome Research Facility (AGRF). As per [5 (link)], RNA-Seq paired-end reads were trimmed for low-quality base-calls (≥ q30) and Illumina adapter sequences using Cutadapt v1.1 [84 ] (parameters: --quality-cutoff 30 --overlap 10 --times 3 –minimum-length 25) with reads trimmed to less than 25 bp discarded and remaining reads sorted into pairs and singleton reads. RNA-Seq reads were then mapped to the Fom-5190a genome assembly via Tophat2 (TopHat2 v2.0.9) (parameters: --b2-very-sensitive -r 80 --mate-std-dev 40 -i 20 -I 4000 -g 20 --report-secondary-alignments --report-discordant-pair-alignments -m 0 --min-coverage-intron 20 --microexon-search --library-type fr-firststrand) [30 (link)]. BAM files generated from Tophat2 output were sorted using SAMtools version 0.1.19 [85 (link)] and visualised using IGV (version 2.3) [86 (link)]. Trimmed sequencing data is available from the NCBI/GenBank database under BioProject number PRJNA294248 (http://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA294248).

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Thatcher L.F., Williams A.H., Garg G., Buck S.A, & Singh K.B. (2016). Transcriptome analysis of the fungal pathogen Fusarium oxysporum f. sp. medicaginis during colonisation of resistant and susceptible Medicago truncatula hosts identifies differential pathogenicity profiles and novel candidate effectors. BMC Genomics, 17, 860.

Publication 2016

Agilent 2100 Bioanalyser Plant Nano system HiSeq platform

Genome Intron Library Plant Rna seq

Corresponding Organization : Commonwealth Scientific and Industrial Research Organisation

Other organizations : University of Western Australia

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RNA integrity was confirmed using the Agilent 2100 Bioanalyser Plant Nano system (Agilent Biotechnologies)
Stranded Illumina TruSeq libraries were generated from 1 μg of total RNA
RNA-Seq paired-end reads were trimmed for low-quality base-calls (≥ q30) and Illumina adapter sequences using Cutadapt v1.1 (parameters: --quality-cutoff 30 --overlap 10 --times 3 –minimum-length 25) with reads trimmed to less than 25 bp discarded and remaining reads sorted into pairs and singleton reads
RNA-Seq reads were then mapped to the Fom-5190a genome assembly via Tophat2 (TopHat2 v2.0.9) (parameters: --b2-very-sensitive -r 80 --mate-std-dev 40 -i 20 -I 4000 -g 20 --report-secondary-alignments --report-discordant-pair-alignments -m 0 --min-coverage-intron 20 --microexon-search --library-type fr-firststrand)

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