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changes.mady.by.user Noa Wigoda

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changes.mady.by.user Dena Leshkowitz

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  1. Quality control: Reads are trimmed using cutadapt (DOI: 14806/ej.17.1.200) (with the parameters --times 2 -q 20 -m 25). In this process, primers corresponding to the Tru-seq protocol are removed.
  2. Quality control: Reads quality control is evaluated using FastQC (with the parameter --cassava). A report file, containing quality results for all of the samples is generated using multiQC.
  3. Mapping to genome: The quality trimmed reads a mapped to Mouse/Human genomes: /shareDB/iGenomes/Mus_musculus/UCSC/mm10/Sequence/Bowtie2Index/genome /shareDB/iGenomes/Homo_sapiens/UCSC/hg19/Sequence/Bowtie2Index/genome, /shareDB/iGenomes/Homo_sapiens/UCSC/hg38/Sequence/Bowtie2Index/genome respectively, using Bowtie2 (doi: 10.1038/nmeth.1923.) (with the parameters --local for all analyses and, in addition, -X 2000 for paired end analyses). Refseq annotation is provided for the mapped genes.
  4. Following the alignment, duplicated reads are removed using samtools view (with the parameters -h -F 4 for all reads and, in addition, -f 0x2 for paired end reads). The remaining unique reads are indexed and sorted using samtools index and samtools sort
  5. Generate statistics on the alignment using flagstat.
  6. Visualization in graphs: The reads are graphically visualized using ngsplot (with the parameters -G -R genebody -C -O samples -D refseq -L 50000).
  7. Peak calling: Significant chip regions (peaks) are evaluated and compared to control samples if present using macs2 callpeak (with the parameters --bw 300 -B -f --SPMR -g -keep-dup auto -q 0.01 for all analyses, BAMPE --nomodel for paired end analyses, and BAM for single end analyses).
  8. Convert to BigWig format: Files containing the predicted peaks coordinates in BedGraph format are converted to BIgWig format using bedtools slop (with the parameters -g -b 0), bedClip stdin and bedGraphToBigWig (with default parameters)
  9. Peak annotation: The predicted peaks are then peaks were merged using FILL IN and were annotated according to the corresponding genome using Homer (with default parameters).

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