Traditional Chinese medicines (TCMs) have been an integral part of Chinese culture and the primary medicinal treatment for a large portion of the population for more than 3000 years 
. Outside of Asia there has been, in recent decades, a growing use of TCMs where they are being taken in conjunction with, or as an alternative to, conventional Western medicine 
. The increasing popularity of TCM products has seen the monetary value of the industry increase to hundreds of millions of dollars per annum
, its growth paralleled by the global increase in the use of complementary and alternative medicines. Despite its increased uptake, the therapeutic benefits of only a small number of TCM products have been scientifically validated 
, with their perceived efficacy being based largely on long-standing beliefs 
Chinese herbal medicines often contain numerous different plant and animal-derived products that combine to act synergistically to affect a desired outcome 
. However, due to the proprietary nature of TCM manufacture, coupled with a lack of industry regulation, the biological origin of contents can be difficult to determine with confidence, leading to questions regarding TCM quality, efficacy and safety 
. Undeclared or misidentified TCM ingredients and adulterants can pose serious health risks to consumers 
. These include: allergenic substances 
, plant toxins 
, heavy metals such as mercury, lead, copper and arsenic 
, and pharmaceutically active compounds of undetermined concentration 
. In the early 1990s the misidentification of the toxic herb Aristolochia fangchi
for the anti-inflammatory agent Stephania tetrandra
led more than a hundred women to suffer kidney failure, with many later developing cancer of the urinary system 
In addition to safety concerns, issues of legality also surround TCMs. These concerns fall into three main categories: matters relating to the trade of endangered species; issues pertaining to honesty of food labelling; and adulteration of samples with drugs. Some TCMs contain plant and animal species 
that fall under the jurisdiction of the Convention on International Trade in Endangered Species (CITES). CITES-listed species (see appendicies at www.cites.org
) that have had long-standing associations and use within TCM include: Asiatic black bear (Ursus thibetanus
, Appendix I listed), Saiga antelope (Saiga tatarica
, Appendix II listed), rhinoceros (all species, Appendix I listed), and non-cultivated varieties of the plant genus Panax
; P. ginseng
and P. quinquefolius
, (Appendix II listed) 
. The CITES appendices include lists of species afforded different levels or types of protection from over-exploitation. Appendix I species are deemed the most endangered and threatened with extinction, with Appendix II and III listed species regarded to be at lower, but still significant, threat levels 
. With an increased international demand for TCMs, ascertaining the biological origins, and hence the CITES status, of ingredients contained variously in capsules, powders, liquids, and tablets represents a complex problem for customs officials. The second issue of legality concerns the mislabelling of TCMs. This might be done intentionally in order to reduce manufacturing costs, or to circumvent customs' scrutiny, or inadvertently if the TCM practitioner unwittingly uses a misidentified product 
. For CITES member states to enforce legislation and to prosecute cases of illegal trade, reliable methods of species identification are needed 
. Lastly, a number of TCM products appear to have been intentionally adulterated with drugs of known pharmacological activity such as anti-hyperglycaemic agents (anti-diabetic medication) and corticosteroids 
, presumably as a means to increase their efficacy.
To date, many of the analyses and identification of botanical components in TCM products have employed chromatographic methods 
. However, these methods may not be able to identify animal species, or be able to uncover all of the ingredients within heterogeneous samples. DNA technology has the potential to provide information about species composition and the honesty of ingredient declarations. For the identification of botanical constituents used in TCMs, the genetic techniques employed include fragment length polymorphism analysis, dot-blot hybridization, micro-arrays, and sequencing of plastid DNA genes 
. Likewise, genetic identification of animal species commonly involves DNA sequencing and characterisation of mitochondrial DNA (mtDNA) genes 
. Despite the variety of genetic work that has been conducted to date, investigative research seems to have focused on detecting the DNA of specific targets within TCMs 
or herbal teas 
rather than investigating all
of the contributing species within a sample simultaneously.
The advent of Second Generation, high-throughput sequencing (HTS) platforms have enabled the rapid sequencing of genes, genomes and metagenomes 
. The ability of these technologies to deep-sequence both PCR amplified plastid and mtDNA markers (using molecular identifier [MID] tags) has allowed the species composition of a variety of complex substrates including faecal material 
, sediments 
and even, in a forensic context, microbial communities on computer keyboards 
, to be determined. The application of HTS technologies to analyse complementary medicines has not been previously attempted, but is likely to prove to be the best approach by which to genetically audit the species composition of multiple TCM samples in parallel.
Given the worldwide popularity, growing use and increasing financial significance of TCMs, an effective means of evaluating these medicines is urgently needed – a sentiment echoed by strategy reports from the World Health Organization (WHO) 
. This study sets out to explore the probative value of HTS approaches by generating species audits from 15 TCMs (; ) seized by border protection officials upon entry into Australia.
Photographs of four TCM samples genetically audited in this study using high-throughput sequencing.
Analysed TCM samples, including sample ID, brand names, and listed package ingredients.