SPADA Draft Documents

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Table 1. Summary of recommendations for PCR primer design

Design Stage

Item

Recommendation

Reason

Preparation for Design Inclusivity Database

Database and literature research on target sequences Full-length genomes or genes

Consensus design to conserved regions

Inclusivity Database Exclusivity Database

fragments lead to design bias

near-neighbor sequences

Sequences likely to cause a false positive due to sequence relatedness or similar symptoms reduce false positives from human genome, human refseq, human microbiome, soil microbes, etc. Needed for proper physical modeling and design

Background Database

Gather contaminating genomes

Reaction Conditions

Gather enzyme, buffer and salts, [NTPs], [Primers, Probes] Save all user settings and input, scoring terms and weights Capture all software parameters

Software Input

Run Software

Save input file

Allow for design to be reproduced, capture input for future meta-analysis using A.I. Allow for design to be reproduced, reduce input errors in subsequent runs Find signature regions that are conserved in the inclusivity and not found in exclusivity Not applicable to long genomes and to many genomes

Run from input file

software can be run from input file

Algorithm

k-mer analysis

Use to find signature regions using inclusivity and exclusivity databases Do not use for finding signature regions Align design regions of all members of inclusivity Target secondary structure, primer hybridization, primer dimers Scores for non-thermodynamic considerations Use thermodynamics-based scanning of primers against genomes Determine how well primers cover all members of inclusivity

Sequence Alignments Sequence alignments

Use for reality check of consensus region only

Physical Chemistry Modeling

Naïve 2-state Tm is not sufficient

Heuristic Scoring

Examples: G-quartets, low complexity, amplicon length, amplicon folding, etc. Check if primers are specific (PrimerBLAST, ThermoBLAST, ThermonucleotideBLAST) Determine mismatches and amount bound for all members of inclusivity Combine thermodynamic, heuristic, kmer, specificty, and other terms into a total score. Don't want control to interfere with desired target(s)

Specificity scoring

Coverage Scoring

Total Scoring

Weighted scoring equation

Add positive control

Check for compatibility of singplexes with the control gene

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