Whole-genome shotgun metagenomic sequencing can accurately identify the presence of all microbial species in a sample without the need for prior knowledge of their genetic sequences.

Whole‑genome shotgun metagenomics sequences every piece of DNA in a sample, so it can discover many species even if they have never been studied before. However, the reads still have to be matched to known genomes in a database; without reference sequences the assignment is uncertain or impossible. In practice, rare or novel organisms may be missed or misidentified, and the accuracy depends on the quality and completeness of the reference database. For example, a sample that contains a previously unknown bacterium will produce reads that cannot be matched to any known genome, so that species will not be reported even though its DNA was sequenced. Thus, shotgun sequencing is powerful but still requires some prior knowledge of genetic sequences for reliable identification.