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Genes (Basel). 2019 May 7;10(5). pii: E343. doi: 10.3390/genes10050343.

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

Author information

1
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 102488, China. zheng.x.s1987@163.com.
2
Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of ChineseMateria Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China. zheng.x.s1987@163.com.
3
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 102488, China. max_liucs@263.net.
4
Guangdong Provincial Key Laboratory of New Drug Development and Research of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, China. zheng.x.s1987@163.com.
5
Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of ChineseMateria Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China. lwu@icmm.ac.cn.
6
Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of ChineseMateria Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China. zhixiangliu88@163.com.
7
Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of ChineseMateria Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China. swjs082lbs@126.com.
8
Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of ChineseMateria Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China. yhshi@icmm.ac.cn.
9
Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of ChineseMateria Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China. xwli@icmm.ac.cn.
10
Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of ChineseMateria Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China. jxu@icmm.ac.cn.
11
Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of ChineseMateria Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China. slchen@icmm.ac.cn.

Abstract

DNA barcoding has been used for decades, although it has mostly been applied to somesingle-species. Traditional Chinese medicine (TCM), which is mainly used in the form ofcombination-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 pastfew years, but conventional NGS technology is hampered in application due to its short sequencingreads and systematic errors. Here, a novel method, Full-length multi-barcoding (FLMB) vialong-read sequencing, is employed for the identification of biological compositions in herbalcompound formulas in adequate and well controlled studies. By directly sequencing the full-lengthamplicons of ITS2 and psbA-trnH through single-molecule real-time (SMRT) technology, thebiological composition of a classical prescription Sheng-Mai-San (SMS) was analyzed. At the sametime, clone-dependent Sanger sequencing was carried out as a parallel control. Further, anotherformula-Sanwei-Jili-San (SJS)-was analyzed with genes of ITS2 and CO1. All the ingredients inthe samples of SMS and SJS were successfully authenticated at the species level, and 11 exogenousspecies were also checked, some of which were considered as common contaminations in theseproducts. Methodology analysis demonstrated that this method was sensitive, accurate andreliable. FLMB, a superior but feasible approach for the identification of biological complexmixture, was established and elucidated, which shows perfect interpretation for DNA barcodingthat could lead its application in multi-species mixtures.

KEYWORDS:

DNA barcoding; ITS2; Sheng-Mai-San (SMS); multi-species mixtures; next-generation sequencing; single-molecule real-time (SMRT)

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