version 0.1.0

This release corresponds to the version that was described in the pre-print Froehlich & Uyar et al on Biorxiv.

0.0.2

News for version 0.0.2

• Now support both insertions and deletions
• Integrated Rmarkdown reports with interactive plotly figures
  • One report per amplicon
• Integrated Rmarkdown reports for pairwise comparison of samples to discover sites that show differential peaks of indel efficiencies in a case-control setting.
• Folders bed and bedgraph replaced with folder indels, which contains:
  • bedgraph files for insertions, deletions, and indels (combined insertions/deletions)
  • bed files for top insertions, deletions (sorted by read support per genotype)
  • indel stats at per-base resolution as a raw tsv file
  • indel stats at expected cut sites
• reports folder now contains one report per each amplicon. Also a sub-folder called comparisons is produced if a comparisons.tsv file is provided, which contains pairwise samples that a user desires to compare (see sample_data/comparisons.tsv file as an example).
• The pipeline now uses R libraries (GenomicAlignments) to extract indel stats from bam files rather than using samtools mpileup.

0.0.1

The pipeline currently supports single-end reads.

Inputs

1. Sample sheet (.csv format) consisting of 4 fields

• sample_name: unique name of the sequenced sample
• amplicon: name of the targeted amplicon (seqname)
• reads: name of the zipped fastq file of the sequenced sample
• sgRNA_ids: Column (:) separated list of the ids of guide RNAs used to target the amplicon

2. Settings file (.yaml format)

• For each amplicon (as identified in the sample sheet)
  • Fasta format sequence of the amplicon
  • Cutsites (text file with sgRNA id and position of expected cut site)
• Location of the sample sheet
• Folder containing the fastq files

Rules

• Quality improvement and control (trimmomatic + fastqc + multiqc)
• Mapping the reads against the amplicon sequence (using bbmap to discover short + long indels)
• Extraction of variants (samtools mpileup + custom R scripts)

Output

• bbmap_indexes: Contains the indices generated for each amplicon sequence
• trimmed_reads: Reads trimmed for quality/adapter sequences using trimmomatic
• aln: contains the BAM files and samtools stats output for each sample's alignment (a sub-folder is created for each amplicon)
• fastqc: FASTQC output for each sample (a sub-folder is created for each amplicon)
• logs: Log files for each task
• multiqc: MULTIQC output
• bedgraph:
• bed: BED files for the location of detected deletions, a TSV file for deletion counts, a PDF file containing plots for the distribution of deletions w.r.t length of deletions.
• bedgraph: BEDGRAPH files for deletion score profiles of each sample (i.e. percentage of reads overlapping a deletion at single-base resolution), TSV file for cutting efficiency of sgRNA cut sites (percentange of reads with deletions at the speicified cut-sites)