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Figure 3: Sieving of sequences after cloning of PCR products obtained with novel primer pairs for target genes nirA, nosZ, and nifH. The 5-step refinement procedure included the removal of (1) low-quality sequencing reads, (2) empty vector sequences, (3) translation products with stops, (4) redundant peptide sequences, and (5) peptide sequences outside the targeted protein family. Where the number of retained sequences decreased steeply indicates potential sources of error in practical applications of the primer pairs.