1. Clone the repository.

   git clone https://github.com/Kkulkarni1/CAPG.git

2. Compile CAPG. The executable is called capg_wgs. It will appear in the CAPG/src/wgs directory.

   cd CAPG/src/wgs
   cmake .
   make

3. Install CAPG. Copy the executable to wherever you need it. If you have root privileges, you can install it into the system path, for example:

   sudo cp capg_wgs /usr/local/bin

The software requires multiple input files.

1. Fasta files containing subgenomic references, one subgenome per file. Pass them in via the --fsa_files command-line option.

2. SAM files containing the reads aligned to each subgenomic reference separately. Pass them in via the --sam_files command-line option.

3. SAM file containing the alignments of selected target regions in each subgenome to each other. Pass it in via the --geno command-line option.

It also requires one command-line option.

You must name the target regions to genotype, including the chromosome name and the start and end positions relative to the whole chromosome. For example, chr1:1-10 means you want to genotype from position 1 to 10 (1 based) in chromosome 1. Use ':' to seperate chromosome name and region index. Use '-' to seperate start and end positions. Pass these in by the --ref_names command-line option.

Please run ./capg_wgs -h for detailed information about all available options.

All the files used and created in this tutorial are in the data folder. In this example we will genotype positions 1 to 5000 of both subgenomes assuming they are homoeologous. Finally, we store the output in VCF files, whose names are provided via the --vcf_files option.

From the src/wgs directory, the command line for genotyping is:

./capg_wgs --ref_names Genome_A:1-5000 Genome_B:1-5000 --sam_files ../../data/aln0A.sam ../../data/aln0B.sam --fsa_files ../../data/refA.fa ../../data/refB.fa --geno ../../data/ref.sam -equal --vcf_files ../../data/A.vcf ../../data/B.vcf

The positions with no coverage in the first genome will not be outputed.

If you have any problems with this software, please contact:

Roshan Kulkarni (roshank@iastate.edu) or Karin S. Dorman (kdorman@iastate.edu)

We also have a similar software for genotyping amplicon sequences, here we briefly mention how to use it.

Installation

Compile CAPG for amplicon. The executable is called capg_amp. It will appear in the CAPG/src/amplicon directory.

   cd CAPG/src/amplicon
   make capg_amp

Command-Line Options

CAPG_AMP(1)

NAME
    capg_amp - genotype tetraploids

SYNOPSIS
    capg_amp --sam_files SAM1 SAM2 --fasta_files FSA1 FSA2 --ref_names REF1 REF2
        [[--genotype_by_clustering [--alignment FILE1 FILE2]]
        [--sample INT --min-subgenomic-coverage FLOAT]
        [--min INT --max INT --expected-errors FLOAT --indel INT --loglik FLOAT
         --min-posterior FLOAT --secondary --soft-clipped INT]
        [--coverage FLOAT --biallelic FLOAT --equal_coverage_test [FLOAT1 FLOAT2]]
        [--drop INT --amplici EXE [--amplici-f FILE --amplici-o STRING --amplici-l FLOAT]]
        [--error_file|--error_data FILE] ...]

DESCRIPTION
    capg_amp genotypes allotetraploids using reads in SAM1 and SAM2 aligned to
    REF1 and REF2 references from fasta files FSA1 FSA2.
    SAM files typically contain reads from a single individual, genotype, or
    accession aligned to multiple amplified targets, but capg_amp genotypes
    one individual at one amplicon.

OPTIONS

Input (required):
    --fasta_files FILE1 FILE2
        Subgenomic reference fasta files (Default: none).
        DEPRECATED: see --ref_fasta_files
    --ref_fasta_files FILE1 FILE2
        Subgenomic reference fasta files (Default: none).
    --sam_files FILE1 FILE2
        SAM files with reads aligned to each subgenome (Default: none)
    --ref_names STRING1 STRING2
        Names of subgenomic reference target regions (Default: none)
    --ref_alignment FILE
        SAM file containing alignment of references (Default: none)

Output (optional):
    --display_alignment
        Display alignments in stderr output (Default: no).
    --vcf_files FILE1 FILE2
        Genotyping output in one vcf file per subgenome (Default: none).
    --subref_fasta_files FILE|FILE1 FILE2
        Subsetted reference regions output to these FASTA files (Default: subsetted_refs[12].fsa)
    --gl
        Toggle GL output to vcf files (Default: yes).
    --name STRING
        Name of accession/individual/genotype; used in vcf header (Default: sample).

Estimation/Inference (optional):
    --genotype_by_clustering
        Genotype by clustering (Default: no).
        Requires command-line arguments --amplici and --clustalo or --mafft.
    --clustalo FILE
        The clustal omega executable (Default: (null)).
    --mafft FILE
        The mafft executable (Default: (null)).
    --alignment FILE1 FILE2
        Alignment input FILE1 and output FILE2 (Default: selected_haplotypes.fa selected_haplotypes.co.fa)
    --misalignment_rate FLOAT
        Maximum allowed subgenomic misalignment rate in [0, 0.5) (Default: 0.30).
        Tolerated proportion of reads from one subgenome aligning to the other.

Screening paralogs and other contaminants (optional):
    -p, --drop INT
        Drop reads aligning to paralogs; -1 to automate (Default: -1)
        Drop INT of four most abundant haplotypes if specified.
        Requires command-line argument --amplici.
    --amplici EXE
        The amplicon denoiser software (Default: none).
        Writes auxiliary files "amplici.fastq" "amplici.fa", and "amplici.out".
        See https://github.com/DormanLab/AmpliCI for more information.
    --amplici_fastq FILE
        Selected reads output to this FASTQ file for denoising (Default: amplici.fastq).
    --write-fastq [FILE]
        Write fastq file for AmpliCI and quit (Default: no).
        See --amplici_fastq to name the file.
        With optional argument write named fastq file after AmpliCI paralog filtering (Default: none).