A sequence reference library is not a prerequisite, but it is something better than nothing
Soil sample source tracking using DNA metabarcoding (or any other methods) is based on a set of data (here considered a local library) and operation taxonomic units (OTUs) instead of species names are used. This means that a universal reference sequence library is not necessary for forensics. As exemplified in this study, results based on the total OTUs came to the same conclusion as that based on the annotated OTUs. However, if the OTUs were annotated to species, genera or families, extra information such as morphological characters could be used and the evidences would be more solid.
To annotate the OTUs, a well-curated sequence reference library is indispensable. The reference library helps to exclude data of experimental artifacts (such as chimeras) and non-target species. Although some efforts have been made, the DNA barcode reference library is still far from being satisfactory due to low species coverage (Lou et al., 2010; Ratnasingham & Hebert, 2007; Tnah et al., 2019), especially for lower plants. For example, there are about 8397 known species worldwide and only 889 species have their rbcL sequences deposited in GenBank (4116 accessions. accessed on Jan. 9, 2021,rbcL in plastid genomes were not considered.).