The statistical power of k-mer based aggregative statistics for alignment-free detection of horizontal gene transfer

Alignment-based database search and sequence comparison are commonly used to detect horizontal gene transfer (HGT). However, with the rapid increase of sequencing depth, hundreds of thousands of contigs are routinely assembled from metagenomics studies, which challenges alignment-based HGT analysis by overwhelming the known reference sequences. Detecting HGT by k-mer statistics thus becomes an attractive alternative. These alignment-free statistics have been demonstrated in high performance and efficiency in whole-genome and transcriptome comparisons. To adapt k-mer statistics for HGT detection, we developed two aggregative statistics T_sum ^S and T_sum ^*, which subsample metagenome contigs by their representative regions, and summarize the regional D₂ ^S and D₂ ^* metrics by their upper bounds. We systematically studied the aggregative statistics’ power at different k-mer size using simulations. Our analysis showed that, in general, the power of T_sum ^S and T_sum ^* increases with sequencing coverage, and reaches a maximum power >80% at k = 6, with 5% Type-I error and the coverage ratio >0.2x. The statistical power of T_sum ^S and T_sum ^* was evaluated with realistic simulations of HGT mechanism, sequencing depth, read length, and base error. We expect these statistics to be useful distance metrics for identifying HGT in metagenomic studies.