![]() IMPORTANCE Taxonomic assignment, assigning sequences to specific taxonomic units, is a crucial processing step in microbiome analyses. Standalone genome cover and integration into Woltka are available:. We present Zebra, a method to compute and threshold the genome cover metric across like samples, a recurrence to estimate genome cover and confirm saturation, and provide guidance for choosing cover thresholds in real world scenarios. We note that it is a threshold on saturated genome cover, not genome cover itself, which indicates a spurious reference hit or distant relative. By compositing the metric across like samples, genome cover saturates even for rare species. We assuage this worry with examination of a large human fecal data set. Genome cover may not saturate for low abundance or low prevalence bacteria. We next introduce saturated genome cover, the true fraction of a reference genome overlapped by sample contents. aureus and true contaminants from the false artifacts of reference overlap. We show how to dynamically predict genome cover by read count and examine our model in Staphylococcus aureus monoculture. We introduce genome cover, the fraction of reference genome overlapped by reads, to distinguish these artifacts. The resulting artifacts create both the illusion of co-occurrence and a long tail end of extraneous reference hits that confound interpretation. ![]() With the Web of Life (WoL) reference database hosting 10,575 reference genomes and growing, the percentage of ambiguous reads will only increase. ![]() Assigning taxonomy remains a challenging topic in microbiome studies, due largely to ambiguity of reads which overlap multiple reference genomes. ![]()
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