Amelia

What to do

A Nextflow pipeline is used to analyze Targeted NGS of Mycoplasma genitalium. The outputs include QC, amplicon statistics, SNP calling, amino acid variation detection, etc.

Prerequisites

Nextflow is needed. The detail of installation can be found in https://github.com/nextflow-io/nextflow.

Python3 is needed. And the pacakage "biopython" is also needed.

Singularity/Apptainer is needed. The detail of installation can be found in https://apptainer.org/

Recommended conda environment installation

conda create -n AMELIA -c conda-forge python=3.10 biopython

conda activate AMELIA

How to run

1. put your data files into directory /fastqs. Your data file's name should look like "JBS22002292_1.fastq.gz", "JBS22002292_2.fastq.gz"

2. put your primer information file in bed format into directory /primers.

3. open file "parames.yaml", set the parameters.

4. get into the top directory of the pipeline and then run command below:

   If you use SLURM, run

   sbatch amelia.sh

   If not use SLURM, run

   bash ./amelia.sh

Result

In the final report, except for some columns related to sequencing quality another 7 columns were added. The each column is named by primer name. In each primer column, the outputs are Absolute matching read counts per amplicon, depth per amplicon, Percentage coverage per amplicon, SNPs per amplicon. These outputs are separated by comma. For example, one primer column lists below:

3548,733.1,58.21,T191144A|ATG:AAG|M:K;T191801C|CTC:CCC|L:P

It means that in the amplification region mapping reads are 3548, depth is 733.1, percentage coverage is 58.21, the nucleotide T changes to A at site 191144, (if the SNP occurs at CDS region) the triplet ATG changes to AAG, the amino acid M changes to K; the second SNP is T to C at site 191801, triplet changes from CTC to CCC, amino acid changes from L to P.

If no SNPs occur at an amplification region, "None" will be added to the output. For example,

2977,609.2,58.21,None

If SNPs do not occur at CDS region, such as rRNA, no triplet and amino acid will be outputted. For example,

9,1.5,45.82,A173799G

Note: some sample data files can be found in directory /fastqs/test_sample. A sample of primer bed file can be found in /primers.