![]() ![]() Replicates were averaged, and genes with FDR adjusted p value <0.05 and fold change was calculated. Algorithms for the sensitive detection of so called low-frequency variants supported only by a small fraction of mapped reads complete the detection tools. Statistical analysis of differentially expressed genes was carried out with the Empirical analysis of Digital Gene Expression data tool in CLC Genomic Workbench. median coverages for nanopore reads were rarely above 5, and were also low for pyrosequencing data (Figure S27). Further, the assembly of millions and billions of RNA-seq reads to. With the CLC Genomics Workbench aligner, 95 of pyrosequencing reads and about 6080 of the nanopore 2D reads pass were mapped (Figure S26 A absolute numbers of mapped loci, see Figure 4 B). RPKM values were calculated for each gene to quantify absolute expression. Although becoming cheaper, transcriptome sequencing still remains an expensive endeavor. The aligned reads were obtained using the RNA-Seq Analysis Tool of CLC Genomics Workbench. The reads of one representative of each of the eight species were imported into CLC Genomics Workbench (GW) v9.5.3 (CLC Bio-Qiagen, Aarhus, Denmark), and low. Bases with low quality were trimmed and reads were mapped to the reference genome, Mycobacterium tuberculosis H37Rv (NCBI Reference Sequence: NC_000962.3). The sequencing of the cDNA libraries was performed on the Illumina NextSeq 500 platform (Illumina, San Diego, CA) using the high output 1X75 cycles configuration.ĬLC Genomics Workbench 9.0.1 version ( Qiagen) was utilized for RNA-seq analysis.ĭe-multiplexed fastq files from RNA-Seq libraries were imported into the CLC software. RRNA was removed, and the cDNA library was prepared using illumina library prep kit. The cultures were harvested by centrifugation and total RNA was extracted using TRIzol® LS reagent (ThermoFisher Scientific) and bead beating followed by extraction with the RNeasy® Mini Kit (Qiagen). ![]() tuberculosis H37Rv was grown to OD595 to ~0.4 in 7H9-Glycerol-Tween-OADC media in tissue culture flasks (50 mL each) and pooled. After 4 h incubation at 37 ☌ with shaking. The final concentration of DMSO was kept constant in each flask. Variants can be SNV, MNV, replacement, insertion, or deletion. Variant track: Displays information on allele variants at the base pair level. Reads track: Displays how all of the (mapped) reads map to the reference genome. The cultures (10 mL) were redistributed into flasks containing each compound (10x MIC) or compound combination or vehicle (DMSO) control. Tracks (see page 536 of CLC GWB manual) Sequence track: Displays the chromosomes of the reference genome. GEO help: Mouse over screen elements for information.
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