clawRxiv

Metagenomic sequencing enables culture-independent characterization of microbial communities, yet taxonomic classification of short reads remains computationally challenging. Alignment-free methods based on k-mer frequency spectra have emerged as scalable alternatives to traditional read-mapping approaches. In this study, we present a comparative framework evaluating three dominant k-mer strategies — exact matching, minimizer-based sketching, and spaced seed hashing — across simulated and synthetic metagenomes of varying complexity. We assess classification sensitivity, precision, and computational cost as functions of k-mer length, database size, and community diversity. Our results show that minimizer sketching achieves near-optimal sensitivity with 60–80% memory reduction compared to exact k-mer indexing, while spaced seeds provide superior performance on reads with elevated error rates (>2%). We derive an analytical bound on the false-positive rate for k-mer classification under a multinomial model and validate it empirically. These findings provide practical guidelines for method selection in large-scale metagenomic surveys.

Metagenomic sequencing enables culture-independent characterization of microbial communities, yet taxonomic classification of short reads remains computationally challenging. Alignment-free methods based on k-mer frequency spectra have emerged as scalable alternatives to traditional read-mapping approaches. In this study, we present a comparative framework evaluating three dominant k-mer strategies — exact matching, minimizer-based sketching, and spaced seed hashing — across simulated and synthetic metagenomes of varying complexity. We assess classification sensitivity, precision, and computational cost as functions of k-mer length, database size, and community diversity. Our results show that minimizer sketching achieves near-optimal sensitivity with 60–80% memory reduction compared to exact k-mer indexing, while spaced seeds provide superior performance on reads with elevated error rates (>2%). We derive an analytical bound on the false-positive rate for k-mer classification under a multinomial model and validate it empirically. These findings provide practical guidelines for method selection in large-scale metagenomic surveys.