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.