Whole Sample Next-Generation DNA Sequencing Method: An Alternative to DNA Barcoding

By Casey Schlenker, Jenna Brooks, Kent Oostra, Ryan McLaughlin

A non-targeted approach improves on the standard technique of DNA barcoding, because it is not subject to inherent bias.

Another concern observed in PCR amplification-based barcoding is drastic over-representation of sample constituents that are actually present in quantities far below 1%, possibly even just trace amounts. This creates the perception of adulteration when there are only incidental amounts of material. This incidental DNA can originate from agricultural equipment, manufacturing surfaces or individuals handling the sample. We performed and replicated several experiments to display the advantages of a non-targeted approach over amplification-based methods. Vouchered botanical reference materials (VBRM) were used to simulate a scenario with incidental contamination, which is an area of concern.⁷ Chamomile (Matricaria chamomilla) and lemon balm (Melissa officinalis) were combined in a 99:1 mass ratio to show how a sample with ~1% contamination would appear to traditional DNA barcoding directly compared to our non-targeted metagenomic analysis. A ~1% threshold was used as it is less than the allowable level of background organic material (2%), according to the U.S. Pharmacopeial Convention.8 The results of these experiments were enlightening. The traditional method of DNA barcoding not only over represented the presence of the ~1% “contaminant”, but also identified numerous background incidental species (see Figure 2). In addition to the two included VRBM species, chamomile and lemon balm, eight other species were amplified by universal primers and sequenced. With the non-targeted approach, chamomile was identified with no other “contaminates” appearing in the dataset. The non-targeted method was able to determine the identity of the product without over representing incidental DNA false positives, allowing us to avoid the misinterpretation of a false positive or trace “adulterant”.

Targeted Method, DNA barcoding — Figure 2. Results from an experiment comparing a traditional targeted method, or DNA barcoding using universal primers for amplification with our non-target whole sample sequencing. The sample analyzed was a 99:1 (by mass) ratio of VBRM grade Matricaria chamomila to Melissa officinalis. The ‘Traditional Targeted Method’ column is a list of species that were identified by that method. In addition to the vouchered species composing the samples, eight other false positive species were identified. The ‘non-target’ whole sample method shows only the one identification, the species that made up 99% of the total VBRM mix.

The advantage of using non-targeted whole sample sequencing is that the data are not constrained by a targeted marker. In addition to reducing bias, this opens up the possibility of analyzing mixed or composite samples as well as processed samples. Comprehensively sequencing the entirety of the DNA present in a sample also allows for the discovery of new data applications. Along with the specific goal of identifying unknown samples, this type of sequencing provides large amounts of complex data to use for further research such as biomarker prospecting. Biomarker prospecting can uncover novel genes or gene regions to use as identifiers for individuals or groups of organisms. We continue to discover innovative uses of the large datasets being generated.

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