primers4clades provides a fully automated pipeline for the design of PCR primers for cross-species amplification of novel sequences from metagenomic DNA or from uncharacterized organisms belonging to user-specified phylogenetic lineages. It implements an extended CODEHOP strategy based on both DNA and protein multiple alignments of coding genes and evaluates thermodynamic properties of the oligonucleotide pairs, as well as the phylogenetic information content of predicted amplicons, computed from the branch support values of maximum likelihood phylogenies. Trees displayed on screen make it easy to target primers to interactively selected clades.

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to ensure your results are stored first sequence is taken as the outgroup while drawing the tree

upload a FASTA file, possibly aligned