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. 2019 Mar 4;7:e6570. doi: 10.7717/peerj.6570

Figure 1. Schematic of primer design and primer theoretical melting temperature distribution.

Figure 1

(A) Standard degenerate primer pools are synchronized and have low nucleotide diversity when sequenced on Illumina sequencers. Nucleotides were removed from the 5′ ends of locus-specific portions of oligonucleotide primers to adjust melting temperature and to introduce nucleotide diversity. Nucleotides were removed from the 3′ ends of locus-specific portions of primers to adjust melting temperature only. Sequencing reactions are initiated using the ‘common sequences’ (purple lines) adjacent to the locus-specific regions of the PCR primers. (B) Distribution of theoretical melting temperatures (Tm, °C) for primer pools using standard EMP primers and modified ShortEMP primers. Modified 515F primer pool Tm distribution is not significantly different from the standard 515F primer pool (Wilcoxon-Mann-Whitney test; p = 0.37), while modified 806R primer pool Tm distribution is significantly different from the standard 806R primer pool (WMW test; p < 0.00001).