SMAP design

This is the manual for the component SMAP design of the SMAP package.

SMAP design was created specifically to design primers for amplicon sequencing, in combination with gRNA design from third-party software such as CRISPOR or FlashFry.

SMAP design takes one or more reference sequences (FASTA and GFF) as input and designs non-overlapping amplicons per reference taking target specificity into account.

SMAP design can be combined with gRNA sequences for CRISPR/Cas targetted mutagenesis of the reference sequences. As such, SMAP design overlaps these amplicons and gRNAs, and selects n (user-defined) non-overlapping amplicons with gRNAs according to several criteria such as number of gRNAs covered by the amplicon, specificity and efficiency scores.

SMAP design creates a primer file, gRNA file, GFF file with all structural features, and optionally a summary file and plot, and input files required for downstream analyses using SMAP haplotype-sites or SMAP haplotype-window and SMAP effect-prediction.

A clear illustration of the implementation and use of SMAP design is described in detail in Develtere et al. (2022).

For more information on optimal coverage of the combinatorial design space of multiplex CRISPR/Cas experiments, please see Van Huffel et al. (2022).

Contents:

• Scope & Usage
  • Scope
  • Integration in the SMAP workflow
  • Function of SMAP design
  • Input of SMAP design
  • Guidelines for gRNA design with CRISPOR, FlashFry, or other
  • Commands & options
  • Output
  • Example usage
• Feature Description
  • Definition of reference gene, CDS, amplicons, gRNA, and overlaps
  • Design rules
  • Avoiding polymorphic sites (e.g. SNPs) during amplicon design
• How It Works
  • Workflow of SMAP design
  • SMAP target-selection
  • gRNA filtering
  • Amplicon generation
  • Assignment of gRNAs to amplicons
  • Amplicon ranking
  • Amplicon and gRNA selection
• Examples
  • Illustration of primer and gRNA design
  • Option restrictedPrimerDesign
  • Option preSelectedPrimers