The obvious disadvantage of protein-level sequence classification is the inability to classify reads originating from non-protein-coding genomic regions. Especially when the genomes of the sequenced microbial strains are also contained in the reference database, Kaiju would be less sensitive than nucleotide-level classifiers, which can assess the entire genome, as seen in the HiSeq and MiSeq data sets. However, due to the high density of protein-coding genes in microbial genomes, the probability of overlap between individual sequencing reads and protein-coding genes increases substantially with increasing read lengths. Furthermore, when using paired-end sequencing, the chance of one mate overlapping with a protein-coding gene is higher than for single-end sequencing, which was also shown in our benchmark where longer and paired-end reads had higher sensitivity compared with shorter single-end reads.

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