Precise species detection of traditional Chinese patent medicine by shotgun metagenomic sequencing

Background Current quality control methods for traditional Chinese patent medicines (TCPMs), e.g., microscopy, thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC), cannot detect herbal species composition with adequate precision. To address this issue, more effective detection methods should be explored. Hypothesis/Purpose We hypothesized that shotgun metagenomic sequencing can fulfill the requirements for the species detection of multi-ingredient TCPMs. Methods Longdan Xiegan Wan (LDXGW), once thought to be the chief culprit in aristolochic acid nephropathy (AAN), was selected to establish the method. It was used for both reference and commercial LDXGW samples. The precision authentication of herbal species contained in multi-ingredient TCPM is based on the shotgun metagenomic sequencing of genomic DNA without PCR amplification. Chemical analyses were also conducted as a contrast test. Results Over 100 G of raw data was obtained, and this value represented more than 0.75 billion reads. After assembling and filtering all the reads, a total of 261 contigs were obtained, which belonged to the ITS2, psbA-trnH, and matK regions of the reference and commercial samples. Because the homology of the rbcL region was high, it was not analyzed in the HTS data. Bioinformatics analysis indicated that the ITS2 region, as a DNA barcode, showed the highest identification efficiency. It could successfully detect all prescribed species, including four processed herbal ingredients, in the lab-made reference samples. The commercial samples all met the requirements of the Chinese Pharmacopoeia according to the TLC and HPLC tests. However, the shotgun metagenomic sequencing detected the substitution of Akebiae Caulis (Mutong) in the commercial samples, while the chemical analyses could not distinguish. Conclusion The results highlight that shotgun metagenomic sequencing is a complementary method for the precise species detection of TCPMs.