Computational protocol: The B. subtilis Accessory Helicase PcrA Facilitates DNA Replication through Transcription Units

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Protocol publication

[…] Approximately 750k paired-end Illumina Next-Seq reads per sample were mapped against the genome of B. subtilis strain JH642 (GenBank: CP007800.1) using Bowtie 2 with the—no-mixed option. This option prevents unpaired alignments, such that only reads that aligned uniquely at both ends were mapped []. As discordant mapping was minimal and did not significantly alter the resulting profile, discordant mapping was active. The resulting. sam file was processed by SAMtools, view, sort, and mpileup functions [], to produce wiggle plots. We tested the effect of removing PCR-based and optical duplicates using Picard v1.3 and found that the same gene regions were identified in subsequent analyses, but at a slightly lower signal:noise ratio. Therefore, in the presented data sets, duplicates were not removed. Myc-PcrA ChIP-Seq data and antibody control data were first normalized to input samples (signal in the ChIP sample minus the signal in the corresponding input sample). Normalized antibody control IP (Mock IP) data representing non-specific signal enrichment was subtracted from the normalized ChIP signal. For DnaC ChIP-Seq, ChIP samples were first normalized to inputs (ChIP minus input). The normalized + PcrA DnaC ChIP sample signal was then subtracted from the normalized—PcrA DnaC ChIP signal at each nucleotide position, establishing the differential signal.ChIP-Seq data were quantified as follows: for each gene and intergenic region, the maximal signal, average signal, area under the curve, and area under the curve divided by total gene length (normalized area under the curve) were calculated. Local background was independently determined for regions proximal to oriC (the 0–300k nucleotide region, where background signal is slightly higher due to higher chromosomal copy number), or distal to oriC, by calculating the average maximal signal in ~100 kb regions that were devoid of peaks. Genes containing a maximum signal of more than 5-fold above background were called as peak-containing regions. […]

Pipeline specifications

Software tools Bowtie, SAMtools, Picard
Application ChIP-seq analysis
Organisms Bacillus subtilis, Escherichia coli