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.