AioMinor was implemented in Perl programming language, including a main script for nucletide and amino acid varation frequency in virus genome. It accepts bascalled fastq files derived from Nanopore amplicon sequencing, cleaned/trimmed Illumina amplicon/normal fastq files (single-end or paired-end) with a SARS-CoV-2/other virus genome. By default, AioMinor analyses SARS-CoV-2 based on an NCBI reference genome (NC_045512.2), but the user can also provide customized SARS-CoV-2 or other virus genome as a reference.

Installation:

1. Create an environment with one step

git clone https://github.com/Hiscox-lab/AioMinor/AioMinor.git
cd AioMinor
conda env create -f my_environment.yml
source activate AioMinor 

2. Create an environment step by step

Third party dependencies:

samtools(>=1.9)

bowtie2(=2.4.1)

minimap2(=2.24)

All these third party tool dependencies should be exported to PATH, so that AioMinor can find them.

Perl module dependencies:

Getopt::Long
File::Basename
Data::Dumper
IO::File
Math::CDF
List::Util
Text::NSP
Bio::DB::Sam
Bio::SeqIO

Usage

Please see the details of each parameter by:

Required options:
  -platform           sequencing platform "nanopore" or "illumina".
  -method             method of sequencing Library Preparation "amplicon" or "cDNA".
  -maxins             maximum fragment length of in your amplicon Library with paired-end sequencing.
  -ref                reference genome sequence in fasta file.
  -codingRegion       coding regions in your reference genome.
  -primerbed          primer scheme in bed file.
  -fq                 fastq(.gz) file for single-end sequencing.
  -fq1                fastq(.gz) file for paired-end sequencing mate 1s.
  -fq2                fastq(.gz) file for paired-end sequencing mate 2s.
  -primerbed          amplicon primer information in bed file.

Optional options:
  -samplename         sample name, "alignment" by default.
  -t/-thread          number of threads, 1 by default.
  -minins             minimum fragment length of in your amplicon Library with paired-end sequencing, "50" by default.
  -o                  output path, "./" by default.
  
  -v/-version         Print version information.
  -h/-help            Print help message.\n\n";

Examples:

1. ARTIC Illumina sequencing data

To analyse ARTIC-Illumina amplicon sequencing data with your chosen primer scheme. (The ARTIC primer bed can be found in the "Primerbeds" folder, and reference genome and coding region of SARS-CoV-2 NC_045512.2 can be found in the "References" folder):

perl AioMinor.pl -t 16 -platform illumina -method amplicon -maxins 500 -ref genome.fasta -codingRegion CodingRegion.txt -primerbed nCoV-2019.primer_V3.bed -fq1 example_R1.fastq.gz -fq2 example_R2.fastq.gz -o example_output

2. ARTIC Nanopore sequencing data

To analyse ARTIC-Nanopore amplicon sequencing data with your chosen primer scheme. (The ARTIC primer bed can be found in the "Primerbeds" folder, and reference genome and coding region of SARS-CoV-2 NC_045512.2 can be found in the "References" folder):

perl AioMinor.pl -t 16 -platform nanopore -method amplicon -maxins 1600 -ref genome.fasta -codingRegion CodingRegion.txt -primerbed nCoV-2019.primer_V3.bed -fq example.fastq.gz -o example_output

3. Normal Illumina sequencing data

To analyse Normal Illumina sequencing data with your chosen primer scheme. (The reference genome and coding region of SARS-CoV-2 NC_045512.2 can be found in the "References" folder):

perl AioMinor.pl -t 16 -platform nanopore -method amplicon -maxins 1600 -ref genome.fasta -codingRegion CodingRegion.txt -fq1 example_R1.fastq.gz -fq2 example_R2.fastq.gz -o example_output

Results

The results can be found under the in output path.

1_Syn_NonSyn_aa**

*_entropy.txt file in this folder prvides the raw nucletide varation frequency compared to the consensus genome,inlcuding Transitions and transversions. *_AA.txt file in this folder prvides the raw amino acid varation frequency compared to the consensus genome,inlcuding synonymous and non-synonymous substitution.

2_Syn_NonSyn_filter**

*_filter.txt file in this folder prvides the filtered nucletide varation frequency,inlcuding Transitions and transversions.

3_Syn_NonSyn_filter_aa**

*_AA_all_AA_filtered.txt file in this folder prvides the details of minor and major amino acids at each amino acid site after filtration in 2_Syn_NonSyn_filter. *_AA_all_condon_filtered.txt file in this folder prvides the details of minor and major condons at each amino acid site after filtration in 2_Syn_NonSyn_filter. *_minor_change_filtered.txt file in this folder prvides frequency of synonymous and non-synonymous substitution compared to the consensus genome after filtration.

alignment/alignment.support_oem**

consensus.txt file in this folder prvides consensus genome sequences.

Test data

There a test data obtained from ARTIC (V3) Illumina sequencing of a cell culture sample in the Testdata folder. AioMinor can be tested with this data in the AioMinor directory.

perl AioMinor.pl -t 16 -platform illumina -method amplicon -maxins 500 -ref ./References/genome_NC_045512.2.fasta -codingRegion ./References/CodingRegion_NC_045512.2.txt -primerbed ./Primerbeds/nCoV-2019.primer_V3.bed -fq1 ./Testdata/cell_illumina_R1.fastq.gz -fq2 ./Testdata/cell_illumina_R2.fastq.gz -o cell_illumina_output