We generated strand-specific mRNA-seq libraries (KAPA Biosystems, Wilmington, USA) for 6 biological replicates of control and treatment conditions for both the 1995 An. coluzzii (cyp-2) and 2014 An. coluzzii (cyp-1) genotypes (Additional file: Table S2). These 24 libraries were sequenced on a single lane of HiSeq3000 SR50 at the UC Davis DNA Technologies Core. Poor quality bases and Illumina adapter sequences were trimmed from the raw reads using Trimmomatic version 0.30 [32 (link)], with the following parameters: LEADING:3, TRAILING:3, SLIDINGWINDOW:4:15, MINLEN:36. The trimmed reads were then assessed for general quality using FastQC version 0.10.1 (Babraham Bioinformatics, Cambridge, UK).
Fastqc version 0
Manufactured by Babraham Bioinformatics
Sourced in United Kingdom
FastQC is a quality control tool for high throughput sequence data. It provides a modular set of analyses that can be used to provide a quick overview of whether the data has any problems that you should be aware of before doing any further analysis.
Automatically generated - may contain errors
7 protocols using fastqc version 0
1
Strand-Specific mRNA-Seq Library Preparation
2
Porcine Genome Sequencing and Analysis
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Genomic DNA from 23 Duroc and 24 Landrace boars sequenced on an Illumina GAII (Illumina, San Diego, USA). The sequencing was performed by a commercial sequencing center (Fasteris, Switzerland) according to manufacturer’s protocols. FastQC version 0.10.1 (Babraham Bioinformatics, UK) was used for quality checking, revealing an overall per-base quality ≥30. Pre-processing of reads was done using a custom Perl script written to remove duplicated reads, trim sequencing primer sequence and remove reads shorter than 0.8 of their original length. On average, 15% of reads were filtered using this pipeline and the remaining reads were aligned to the Sus scrofa 10.2 reference genome [55 (link)] using the software Bowtie2 version 2.0.0 with default parameters [56 (link)]. Mapped reads were sorted by their chromosomal coordinates using Samtools version 0.1.18 [57 (link)]. The Picard AddOrReplaceReadGroups program (http://broadinstitute.github.io/picard/ ) was used to assign unique IDs to the files before SNP calling.
3
Transcriptomic analysis of F. columnare
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Adapter removal and quality trimming of FASTQ files was conducted using Trimmomatic [35 (link)] by the HudsonAlpha Genomic Service Lab. Data quality was assessed using FastQC version 0.10.1 (Babraham Bioinformatics). Reads were aligned to F. columnare strain 94–081 [36 (link)] protein-coding sequences (Accession number NZ_CP013992) using the Bowtie2 software [37 (link)]. The alignment statistics were obtained using Samtools [38 (link)]. Differentially expressed genes were identified using EdgeR [39 (link)]. Genes with an FDR-adjusted p value < 0.05, and fold change > 2 were identified as being differentially expressed. Functional annotation and gene ontology were also conducted using BLAST2Go PRO software [40 (link)]. Gene descriptions were annotated as in GeneBank of NCBI. When appropriate, gene annotations were updated with information from InterPro protein database by InterProScan 5 [41 (link)] for predicted functional domains.
4
Whole Genome Sequencing for Mini-MLST
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WGS was used to support the mini-MLST discriminatory ability. The WGS library was prepared using KAPA HyperPrep kits (Roche, Switzerland). The Illumina MiSeq platform was used for WGS, and 250-bp paired-end sequencing was performed. The obtained reads were quality checked using FastQC version 0.11.5 (Babraham Bioinformatics, UK) and assembled using Burrows-Wheeler Aligner version 0.7.17 (33 (link)). Ridom SeqSphere+ (Ridom, Germany) P. aeruginosa seed genome strain PAO1 (NC_002516.2 ) was used as the reference genome. To remove unmapped reads, reads with poor quality, and duplicates, SAMtools version 1.9 was used (34 (link)). After reference mapping, all positions with less than 10% coverage, and all ambiguous positions (less common bases represented at least 10% of bases in the target position) were removed from further analyses. SAMtools version 1.9 was used together with BCFtolls version 1.9 software to obtain consensus sequences (35 (link)). With SeqSphere+ version 7.8 (Ridom, Germany), the genomes were compared with a gene-by-gene approach using an incorporated P. aeruginosa cg-MLST scheme and P. aeruginosa ag-MLST scheme comprising 3,867 cg- and 1,647 ag-genes, respectively (13 (link)). A MST was constructed to visualize the allelic differences between the isolates.
5
Leaf Transcriptome Sequencing and Assembly
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Total RNA was extracted from each leaf sample using the Quick-RNA Miniprep Kit (Zymo Research, USA) as per the manufacturer's instructions and was shipped on dry ice to the Agricultural Research Council Biotechnology Platform (ARC-BTP) in Pretoria, South Africa for sequencing on the HiSeq platform (Illumina Inc., USA). For each sample, the data generated from sequencing was analysed as follows. The read quality was assessed using FastQC version 0.11.5 (Babraham Bioinformatics) and when necessary, Trimmomatic version 0.36 [16] was used to trim. De novo assembly was then performed using SPAdes version 3.10.1 [17] (link) according to the developer's instructions. Nucleotide blast was performed on all contig using BLAST+ [18] .
6
Comprehensive Yeast Genome Sequencing Pipeline
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The general quality of the reads was assessed using FastQC version 0.11.5 (Babraham Bioinformatics) after which reads were mapped to the reference S288c genome (version R64) using bwa-mem version 0.7.12 with default settings (Li and Durbin 2010 (link)). Indels and SNVs were called using GATK version 4.0.0.0 (McKenna et al. 2010 (link)), according to GATK best practice recommendations. Duplicates were marked and HaplotypeCaller was used to call variants while setting ploidy to the level determined by propidium iodide staining. Regions with aneuploidies were later recalled using the adjusted local ploidy. Variants present in the ancestral strain were filtered out, as well as SNVs that were identical in more than three independent samples, and indels identical in more than one independent sample. All of the variants that were filtered out in this way were ambiguous calls, as determined by manual curation. Finally, all remaining SNVs and indels were verified using intensive manual curation.
7
High-Throughput RNA Sequencing Protocol
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Total RNA was extracted from each leaf sample using the Quick-RNA Miniprep Kit (Zymo Research, USA) as per the manufacturer's instructions and was shipped on dry ice to the Agricultural Research Council Biotechnology Platform (ARC-BTP) in Pretoria, South Africa for sequencing on the HiSeq platform (Illumina Inc., USA). For each sample, the data generated from sequencing was analysed as follows. The read quality was assessed using FastQC version 0.11.5 (Babraham Bioinformatics) and when necessary, Trimmomatic version 0.36 [16] (link) was used to trim. De novo assembly was then performed using SPAdes version 3.10.1 [17] (link) according to the developer's instructions. Nucleotide blast was performed on all contig using BLAST+ [18] (link).
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