4.2. Unexpected species detected via shotgun metabarcoding
This study also found several unexpected species in both mock and pharmaceutical samples, e.g.P. quinquefolius , as a common adulterant of P. ginseng , was detected in HSZY192. Then the ITS2 barcode of P. quinquefolius was used as the reference sequence, and the shotgun sequencing reads of pharmaceutical samples were used for short reads mapping using CodonCode Aligner by employing bowtie2 program. The proportion of reads belonging to P. ginsengat the SNP positions in ITS2 were calculated (Supplementary Table 9 ), and the results showed that the reads of P. ginsengwere dominant in A03, HSZY152 and HSZY191, while the reads of P. quinquefolius were dominant in HSZY192. Then, this result was verified by using SNPs positions in the ITS2 region of P. ginseng andP. quinquefolius , and confirmed that P. quinquefolius did exist in HSZY192. The analysis results of the two methods were consistent, which proves that shotgun metabarcoding technology can accurately detect adulterated ingredients of the prescription ingredients. Because P. ginseng and P. quinquefolius were derived from closely related plants (The national pharmacopoeia committee of China, 2020b), their morphological, tissue structure, and chemical composition are very similar (Yap et al., 2008). If they were made into decoction pieces, powdered or traditional herbal patent medicines, it was even more difficult to distinguish each other. In addition, the fungi detected in this study mainly includeFusarium , Cystofilobasidium , Aspergillus , andTausonia at the genus level. These fungal contaminations may occur in multiple stages such as planting, storage, and transportation (Pitt et al., 2013). There are currently more than 20 kinds of herbal materials that need to be tested for mycotoxins in the current Chinese Pharmacopoeia, including Nelumbinis Semen and Coicis Semen, which are the prescription ingredients of SLBZS in this study (The national pharmacopoeia committee of China, 2020a; The national pharmacopoeia committee of China, 2020b). However, the total number of fungal reads detected in this study was very small, accounting for only 0.44% of the total reads in the ITS2 region. In another traditional herbal patent medicine study, the ITS2 sequence of fungi belonging toAspergillus was detected in Longdan Xiegan Wan (Xin et al., 2018a). But this study did not compare the reads number ofAspergillus and prescription ingredients, and it was likely to overestimate the number of fungi presented in LDXGW.