Find Giardia Primer Regions

The Snakemake pipeline aims to find potential giardia primer regions

General Workflow

• Pairwise alignment

  • one of the input sample is set as reference sequence
    • pairwise alignment of each input file against the reference using the all vs all comparison of nucleotide sequences tool nucmer from MUMer (hmmer v3.3)
    • output in delta format
  • the alignments are filtered using the MUMer delta-filter function
    • this script is a wrapper around nucmer that filters 1-to1 alignments
  • extract the filtered alignment information and summarize them in a *.txt
  • combine the alignment information of all samples into an alignment information matrix
  • after sorting the matrix, filter by length and whether the alignment positions can be found in all input samples

• creating multiple sequence alignments (MSA) of the filtered genomic regions

  • extracting the filtered sequence regions per sample in fasta format
  • computing MSAs for each sequence region using MAFFT (v7.471)
  • using a sliding window filter on the MSAs to determine if the input samples can be distinguished by DNA variations within the window
    • can wie finde differentiating regions in the MSAs

• read mapping to determine the allele sequence heterogeneity (ASH) per position and sample

  • map the reads against the initial fasta files per sample using Bowtie2 (v2.4.1)
  • extract specific regions from the bam files using samtools (v1.3)
  • count reads and bases per position using pysamstats (v1.1.2)

• combine the data

  • combine the statistics in a final table
  • rank the MSAs by their statistics and report number of position of interest (POI) per alignment

  Set up

  The pipeline needs Snakemake and Biopython. We recommend to create a Conda environment:

  cd designated/path
  git clone https://github.com/rki-mf1/Find_Giardia_Primer_Regions.git
  cd Find_Giardia_Primer_Regions
  conda create -f pipeline_conda_env.yaml -n GiardiaEnv
  conda activate GiardiaEnv

  Run the Pipeline

  The Pipeline needs a config file (see config_template.yaml) and a samplesheet (see sample_template.txt - a list of the samples and sample names) as input.

  snakemake -s Snakefile --configfile /path/to/config_template.yaml --use-conda