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.