3.3. Ingredients analysis of SLBZS pharmaceutical samplesvia shotgun metabarcoding
There were 84 reliable OTUs were
obtained from the shotgun sequencing data of pharmaceutical samples.
Among them, OTUs for ITS2, psbA-trnH , matK and rbcLwere 46, 14, 13, and 11 were, respectively. There were 50,797 reads have
been mapped to ITS2 OTUs, which far exceeded the number of reads mapped
to psbA-trnH , matK and rbcL .
For ten labeled ingredients, there were detected ten, ten, nine, and
seven ingredients have been detected in A03, HSZY152, HSZY191, and
HSZY192, respectively (Figure 2-3, Supplementary Figure 1-2 ).
Detailed reads of the prescription ingredients in the pharmaceutical
samples based on four barcodes were shown in Supplementary Table
4-7 . When using the ITS2 region for ingredient detection, it was found
that the sequences of W. cocos , N. nucifera , L.
purpureus , W. villosa , and Glycyrrhiza sp. were detected
in the pharmaceutical samples. Among them, Glycyrrhiza sp.represents one of the three original species recorded in the Chinese
Pharmacopoeia “Glycyrrhizae Radix et Rhizoma” item(Supplementary Table 1) , because there were two types of OTUs
identified as Glycyrrhiza sp. , one of which was identified asG. uralensis and the other one as G. glabra or G.
inflata . The ITS2 sequences of A. macrocephala ,Platycodon grandiflorus , C. lacryma-jobi , and Panax
ginseng have been detected in three of the four samples. The sequence
of D. oppositifolia was not detected in all pharmaceutical
samples based on the ITS2 region. Species detection based on thepsbA-trnH sequences revealed
that sequences of D. oppositifolia , N. nucifera , L.
purpureus , W. villosa , and Panax sp. existed in all
pharmaceutical samples. The psbA-trnH sequences ofGlycyrrhiza sp. and Platycodon grandiflorus were detected
in A03, HSZY152, and HSZY191, but not in HSZY192. In contrast, thepsbA-trnH sequence of C. lacryma-jobi was only detected in
HSZY192. The psbA-trnH sequence of A. macrocephala was not
detected in all pharmaceutical samples. The identification results based
on other two chloroplast sequences matK and rbcL were
consistent with the identification results of the psbA-trnHregion, except for D. oppositifolia . D. oppositifolia was
not detected in one of pharmaceutical samples based on the matKregion, while it was detected in all pharmaceutical samples based on therbcL and psbA-trnH regions.
At the same time, sequences of some unlabeled ingredients were also
detected. For example, the ITS2 sequence of Panax quinquefoliuswas detected in HSZY192. Based on the ITS2 sequence, 30 fungal-related
OTUs were obtained from two mock samples and three of the four
pharmaceutical samples, belonging to 14 families and 18 genera(Supplementary Figure 4-5, and Supplementary Table 8) . However,
the overall number of fungal reads detected in this study was relatively
small comparing to reads belonging to labeled ingredients, accounting
for only 0.44% of the total reads of the ITS2 region.