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Open AccessArticle

Full-Length Multi-Barcoding: DNA Barcoding from Single Ingredient to Complex Mixtures

School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 102488, China
Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China
Guangdong Provincial Key Laboratory of New Drug Development and Research of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, China
Authors to whom correspondence should be addressed.
Present Address: Chinese Pharmacopoeia Commission, Beijing 100061, China.
Genes 2019, 10(5), 343;
Received: 2 April 2019 / Revised: 22 April 2019 / Accepted: 29 April 2019 / Published: 7 May 2019
(This article belongs to the Special Issue DNA Barcoding and Metabarcoding of Complex Matrices)
DNA barcoding has been used for decades, although it has mostly been applied to some single-species. Traditional Chinese medicine (TCM), which is mainly used in the form of combination-one type of the multi-species, identification is crucial for clinical usage. Next-generation Sequencing (NGS) has been used to address this authentication issue for the past few years, but conventional NGS technology is hampered in application due to its short sequencing reads and systematic errors. Here, a novel method, Full-length multi-barcoding (FLMB) via long-read sequencing, is employed for the identification of biological compositions in herbal compound formulas in adequate and well controlled studies. By directly sequencing the full-length amplicons of ITS2 and psbA-trnH through single-molecule real-time (SMRT) technology, the biological composition of a classical prescription Sheng-Mai-San (SMS) was analyzed. At the same time, clone-dependent Sanger sequencing was carried out as a parallel control. Further, another formula—Sanwei-Jili-San (SJS)—was analyzed with genes of ITS2 and CO1. All the ingredients in the samples of SMS and SJS were successfully authenticated at the species level, and 11 exogenous species were also checked, some of which were considered as common contaminations in these products. Methodology analysis demonstrated that this method was sensitive, accurate and reliable. FLMB, a superior but feasible approach for the identification of biological complex mixture, was established and elucidated, which shows perfect interpretation for DNA barcoding that could lead its application in multi-species mixtures. View Full-Text
Keywords: DNA barcoding; single-molecule real-time (SMRT); multi-species mixtures; next-generation sequencing; Sheng-Mai-San (SMS); ITS2 DNA barcoding; single-molecule real-time (SMRT); multi-species mixtures; next-generation sequencing; Sheng-Mai-San (SMS); ITS2
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Zhang, P.; Liu, C.; Zheng, X.; Wu, L.; Liu, Z.; Liao, B.; Shi, Y.; Li, X.; Xu, J.; Chen, S. Full-Length Multi-Barcoding: DNA Barcoding from Single Ingredient to Complex Mixtures. Genes 2019, 10, 343.

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