Scope & Usage

Scope

SMAP snp-seq designs HiPlex primers encompassing dedicated polymorphic SNP sites, while avoiding neighboring ‘background’ SNPs. It is a simple application to design primer panels for targeted multiplex amplicon resequencing taking known polymorphisms into account, and can be directed to pre-selected locations like GBS loci or candidate genes.

Input

SMAP snp-seq only requires a reference sequence FASTA file and one VCF file with the SNPs that need to be targeted. Optionally, one may provide a BED file with selected template regions, or a VCF file with ‘background’ SNPs that need to be avoided during primer design. Last, one may create a customized reference for a particular sample set by providing a VCF file with SNPs where the reference nucleotide is substituted by the alternative nucleotide in the reference sequence prior to primer design.

Output

SMAP snp-seq provides a list of primers to order.
SMAP snp-seq creates a BED file with SMAPs to delineate HiPlex loci for downstream analyses with SMAP haplotype-sites.
SMAP snp-seq creates a GFF file with borders to delineate HiPlex windows for downstream analyses with SMAP haplotype-window.
SMAP snp-seq plots feature distributions such as length of amplicons.

Integration in the SMAP workflow

../_images/SMAP_global_scheme_home_snp-seq1.png

SMAP snp-seq is run on a reference sequence FASTA file and one or two VCF files, after variant calling and before SMAP haplotype-sites or SMAP haplotype-window. SMAP snp-seq designs primer panels for HiPlex amplicon sequencing.

Guidelines for variant calling

See Veeckman et al. (2019) for a comparison of different SNP calling methods.

Commands & options

Mandatory options for SMAP snp-seq

SMAP snp-seq only needs a reference sequence and known SNP positions to target.

--reference ### The FASTA file with the reference sequence [no default].

--target_vcf ## The VCF file with SNPs [no default].

Command line options

See tabs below for command line options and specific filter options.

Input data options:

-i, --input_directory ##### (str) ## Input directory [current directory].

--template_region ######## (str) ## Name of the BED file in the input directory containing the genomic coordinates of regions wherein primers must be designed [no BED file provided].

--background_vcf ############## Name of the VCF file in the input directory containing target SNPs [no VCF file with target SNPs provided].

--customized_reference ########## Name of the VCF file in the input directory containing non-polymorphic differences between the reference genome sequence and the samples for primer design [no VCF file with reference genome differences provided].

Amplicon design options:

--maximum_variant_distance ####### (int) ### Maximum distance (in bp) between two variants to be included in the same template region [500].

--flanking_region ########## (int) ### Length of the flanking region (in bp) to be added on both ends of the central template region [half of the maximum variant distance].

--maximum_target_size ############### (int) ### Maximum size (in bp) of a target region [10].

--minimum_target_distance ############ (int) ### Minimum distance (in bp) between two target regions in a template [0].

--minimum_amplicon_size ####### (int) ### Minimum size of an amplicon (incl. primers) in bp [100].

--maximum_amplicon_size ####### (int) ### Maximum size of an amplicon (incl. primers) in bp [110].

--offset ####################### (int) ### Size of the offset at the 5’ and 3’ end of each target region. Variants in the region covered by offset are not tagged as targets for primer design [0, all variants are potential targets].

--minimum_primer_size ######## (int) ### Minimum size (in bp) of a primer [18].

--maximum_primer_size ######## (int) ### Maximum size (in bp) of a primer [27].

--optimal_primer_size ######## (int) ### Optimal size (in bp) of a primer [20].

--maximum_mispriming ###### (int) ### Maximum allowed weighted similarity of a primer with the same template and other templates [12].

--maximum_number_degenerate_nucleotides ## (int) ### Maximum number of degenerate nucleotides (N) in a primer sequence [0].

--region_extension ########### (int) ### Extend template regions in the BED file provided via the --template_region option at their 5’ end 3’ end with the provided value [0, no template region extension].

--retain_overlap ############# Retain overlap among template regions [overlap in template regions is removed].

--split_template_region ######### Split the regions in the BED file provided via the --template_region option in multiple templates based on the maximum_variant_distance [template regions are not split].

Options may be given in any order.

Command to run SMAP snp-seq:

python3 SMAP_snp-seq.py -i /path/to/dir/ --target_vcf variants.vcf --reference genome.fasta

-o, --output_directory ### (str) ### Path to the output directory [current directory].

--border_length ##### (int) ### Border size used in the GFF file that defines the windows for SMAP haplotype-window [10].

--suffix ########## (str) ### Suffix added to output files [set_1].

Options may be given in any order.

Command to run SMAP snp-seq with adjusted border length and suffix to denote the design settings:

python3 SMAP_snp-seq.py -i /path/to/dir/ --target_vcf variants.vcf --reference genome.fasta --border_length 10 --suffix Lp_120_180bp

Example commands

Basic command to run SMAP snp-seq with target SNPs:

python3 SMAP_snp-seq.py -i /path/to/dir/ --target_vcf variants.vcf --reference genome.fasta

Command to run SMAP snp-seq for a set of target SNPs while avoiding background SNPs for primer design:

python3 SMAP_snp-seq.py -i /path/to/dir/ --target_vcf variants.vcf --reference genome.fasta --background_vcf background_snps.vcf

Command to run SMAP snp-seq with a set of SNPs to substitute in a customized reference sequence:

python3 SMAP_snp-seq.py -i /path/to/dir/ --target_vcf variants.vcf --reference genome.fasta --customized_reference reference_variants.vcf

Command to run SMAP snp-seq for a specific set of loci (template regions):

python3 SMAP_snp-seq.py -i /path/to/dir/ --target_vcf variants.vcf --reference genome.fasta --template_region gbs_centralregion.bed

Output

By default, SMAP snp-seq does not provide graphical output.