Improving Marker Gene Sequencing and Analysis Using Curated Single-Gene Databases: nifH as a Case Study

Nucleotide sequence repositories have rapidly grown in recent years due to the accelerating rate at which sequence data is generated. While the breadth of available sequence data is an important resource across many research fields, individual studies often target particular genes, and therefore require only subsets of multi-gene databases. Here, we present: (1) a workflow using existing software to curate comprehensive single-gene databases from multi-gene public repositories, (2) an in silico method for evaluating PCR primer performances, and (3) a software package that combines phylogenetic and sequence identity approaches to link taxonomic identity to marker genes without the need for metagenomic binning. Curated single-gene databases enable these analyses, which when paired with stable isotope techniques, can link biogeochemical processes to the responsible microorganisms. We demonstrate these applications using nifH, the most commonly used marker gene for nitrogen fixation. These approaches can be applied to other genes (e.g., mcrA, amoA), and their value will increase as sequence repositories continue to grow.