2.3. Sanger sequencing, HTS, and data analysis
The PCR products of medicinal materials were bi-directionally sequenced
using an ABI 3730xL DNA Analyzer (Thermo Fisher Scientific Inc., USA).
After constructing a PCR-free library, the meta-genomic DNA of SLBZS was
sequenced using the Illumina NovaSeq platform. For the Sanger sequencing
data, Codoncode Aligner v 9.0.1
(CodonCode Corp., Dedham, MA, USA) was used to assemble the sequencing
chromatograms and remove the primer
sequences. For the Illumina
sequencing data, bioinformatics analysis methods including low-quality
sequence filtering, reads enrichment, sequences assembly, short
sequences mapping, chimera detection and other steps were performed as
described in the previous study (Liu et al., 2021a). Usearch v11 (
https://www.drive5.com/usearch/) was used to cluster sequences
with 100% identity into operational taxonomic units (OTUs), and
representative sequences in each OTU were selected for further analysis.
The sequencing depth and coverage values were calculated using samtools
v1.10 (Etherington et al., 2015). The representative sequence of each
OTU e was used for further analysis when the sequence depth is ≥3 and
the coverage is ≥95%, otherwise, it was removed as a poor quality OTU.
The remaining high-quality OTUs were used for species assignment by
searching the DNA barcoding system for traditional Chinese medicines
(TCM-BOL) (Chen et al., 2014a), the
barcode of life data system (BOLD) (Ratnasingham & Hebert, 2007) and
the nucleotide of GenBank databases (Nt) using the basic local alignment
search tool (BLAST) (Camacho et al., 2009). Finally, the statistics and
taxonomic visualization of the species composition of prescription
ingredients in the SLBZS, were performed using MEGAN v 6.19.4 (Huson et
al., 2016).
2.4. The validation of
adulterant ingredient, P. quinquefolius, in pharmaceutical
samples based on DNA Barcoding and SNP detection technologies
To verify the reliability of the
presence of P. quinquefolius in the pharmaceutical samples,
SNP-based identification method was used for distinguishing the species
of P. ginseng and P. quinquefolius from each other based
on ITS2 sequences (Chen et al., 2013). Specific primers for the ITS2
region of P. ginseng and P. quinquefolius were designed
using Oligo 6 in this study. The forward and reverse primers are
5’-AGTCTTTGAACGCAAGTTG-3’ and 5’-CAAGGACTCGCATTTGGGC-3’, respectively.
Subsequently, powders of P. ginseng and P. quinquefoliusroots were mixed in different ratios of 1: 9, 3: 7, 5: 5, 7: 3, and 9:
1, to verify whether the method has the ability to detect the presence
of P. quinquefolius in the mock mixed powder. DNA extraction, PCR
amplification, and sequencing were performed according to the relevant
methods described in Sections 2.2 and 2.3. Sequence alignment, SNP
detection, and peak map observation were performed using
CodonCode Aligner v 9.0.1. Finally,
this primer pair was used to amply the specific ITS2 regions of P.
ginseng and P. quinquefolius , and to observe whether there were
double peaks in the sequencing traces.