4. Discussion
4.1. Feasibility and advantages of shotgun metabarcoding for the authentication of labeled ingredients in SLBZS
Shotgun sequencing technology has been successfully applied to species detection of complex components in areas such as biodiversity assessment (Crampton-Platt et al., 2016; Warden et al., 2016), animal diet analysis (Srivathsan et al., 2015), and food composition identification (Haiminen et al., 2019). This study used high-throughput sequencing data of total DNA of traditional herbal patent medicine to test whether multiple ingredients could be detected in a single analysis. The results showed that, ten and nine prescription ingredients were successfully detected in lab-made samples using shotgun metabarcoding. C. lacryma-jobiwas not detected in one of the mock samples. We guessed that becauseC. lacryma-jobi was used as a medicinal ingredient after being roasted, and so the DNA might be degraded severely after high-temperature treatment, which made it unsuccessful to obtain these commonly used barcode regions. It might also be due to the different DNA content, inhibitory substances contained in different herbal materials and the difficulty of DNA extraction, resulting in the difference in the number of reads obtained. In the mock sample HSZY159, only the ITS2 sequence of C. lacryma-jobi was obtained, and the number of reads was only 12, and the sequence depth was only 4.89. In the initial data analysis of this study, it was found that D. oppositifolia could be analyzed by chloroplast regions, but the relevant assembly sequence was not obtained in the ITS2 region of the nuclear genome. This study and several other studies had shown that the success rate of ITS2 sequences amplification of D. oppositifolia was very low (Meng et al., 2020). This leads to the lack of its reference sequence in the database, and the relevant reads couldn’t be mapped. To obtain reliable reference sequences, the genomic sequences associated withDioscorea sp. were searched in the SRA public database of NCBI, and the ITS2 region was obtained through the assembly. Finally,D. oppositifolia was successfully detected in two mock samples based on the ITS2 region. In addition, Poria, as a fungal medicinal material, is the sclerotium of W. cocos , in which there is no chloroplast genes. Its ITS2 sequence was not successfully obtained by amplification using universal primers (Chen et al., 2010). However, the ITS2 sequence of W. cocos was successfully found in both mock and pharmaceutical samples via shotgun metabarcoding in this study, and further verified that shotgun metabarcoding could overcome the limitation of amplicon-based methods (Liu et al., 2021a). The positive control of P. quinquefolius added to the mock sample was also successfully detected. The result proved the feasibility and sensitivity of this method. The consistency analysis of the assembled sequences and the reference sequences showed that the method has high accuracy. In addition, due to the problem of uneven mixing when preparing a PCR-free library, there were some differences in sequencing datasets of different samples (Supplementary Table 3 ). Nevertheless, it had little effect on the results, especially for the labeled ingredients. For example, only 3.45 Gb of shotgun sequencing data has been generated for HSZY159, but all the labeled ingredients were detected. The similar results has been reported by study of Qingguo Wan (Liu et al., 2021b), which might due to the result of the abundant copies of chloroplast and ITS2 sequences in the plant cells (Liu et al., 2021b). Therefore, this study provided a reliable and operable method for the identification of biological ingredients of traditional herbal patent medicine.