Untemplated nucleotides search

Pipeline for detection of untemplated nucleotides from RNA-seq data

Requirements

• cutadapt 2.8 with Python 3.8.10
• bowtie 1.2.3
• samtools 1.10
• graphviz 2.43.0
• seqtk 1.3
• BBMap and default-jre
• GPL Ghostscript 9.50
• weblogo 3.7.8

How to run the pipeline on example dataset

cd example
snakemake -j 32 --snakefile ../Snakefile

For dry run without execution you can use

snakemake -j 32 --dry-run ../Snakefile

General recommendations

I recommend you to keep the following directories structure

|-analysis_name
  |
  |-data
  |  |
  |  |-reference*
  |  |-reads*
  |
  |-configs
  | |
  | |-config.yaml*
  |
  |-results
    |
    |--iter_0
    |  |
    |  |-trimmed_reads
    |  |-alignments
    |  | |
    |  | |-mapped
    |  | |-unmapped
    |  | 
    |  |-fastq_from_alignments
    |  | |-mapped
    |  | |-unmapped
    |  | 
    |  |-original
    |    |
    |    |-mapped
    |
    |--iter_1
    |--iter_n

Folders data/reference and data/reads should be preexisting and specified in config file.

Required files

configs/config.yaml must containg all necessary information about pipeline parameters (see example/configs/config.yaml)

data/reference must contain reference genome in fasta format

data/reads must contain reads in fastq format

Other folders will appear during the execution of pipeline.

Outputs

results/iter_*/trimmed_reads contains reads with 1 cuted base from 3' end

results/iter_*/alignments contains all mapped results, including mapped and unmapped alignments in bam format

results/iter_*/fastq contains fastq files extracted from bam

results/iter_*/original contains original non-trimmed reads in fasta fromat and their sequence logos for each length specified in config file

Create pipeline graph

snakemake --dag | dot -Tsvg > dag.svg

Without trimming

With trimming

snakemake --rulegraph | dot -Tsvg > rulegraph.svg