1.2.1. Sequencing Technology

Advances in sequencing technology have radically transformed the potential for DNA barcoding over the last decade by significantly reducing costs and time [6]. The current state-of-the-art sequencing platforms can rapidly sequence tens to hundreds of millions of short-length DNA fragments (50–300 base pairs with Illumina) or tens to hundreds of thousands of long DNA fragments (10,000–30,000 base pairs on PacBio® and Oxford Nanopore). The scale of targeted sequencing projects has expanded such that a single researcher can generate barcode sequences from hundreds or thousands of extracted DNA samples in a matter of hours [7–9]. The expanding scale of sequencing presents a grea<sup>t</sup> opportunity for the barcoding community, as it allows for rapid generation of a universal DNA barcode library across the Tree of Life. This is critical, as high-throughput sequencing leads to a better curated database of barcode sequences from known species, but also a greater representation of sequences from unidentified species (e.g., dark taxa, [10]). The universal library of DNA barcodes from known species is being populated at an increasing pace, but the global scientific community still lacks reference barcode data for a majority of species across all major lineages (Figure 1).

**Figure 1.** Numbers of species and DNA barcodes across the Tree of Life. The number of species in each of the four major groups of organisms on Earth (blue bars) according to the Catalog of Life are given along with the number of published barcode sequences in BOLD (green bars). Inset shows the major green plant clades (blue bars) with the number of barcode sequences in BOLD (green bars) adjacent to the number of accepted species (according to [11]). The estimated percentage of all species with DNA barcode sequences for that group is provided above the bars in this plot.

As the sequencing technology landscape continues to expand (also see Section 3 below), so does the traditional view of DNA barcodes. Longer sequence reads have led some researchers to consider longer barcode sequences with potentially greater discriminatory power for taxonomic identification. A number of recent studies have presented "superbarcodes" [12,13] or "ultra-barcodes" [14,15] as approaches that leverage whole organelle genomes (e.g., the chloroplast) or a combination of organellar and ribosomal DNA to provide significantly longer sequence data for barcoding. The super- or ultra-barcoding approach has been most commonly used for plants, which present a number of challenges to traditional DNA barcoding. Another alternative for traditional DNA barcoding leverages high-throughput sequencing technology to "skim" the genome (e.g., genome skimming, low-coverage sequence reads from a whole genome) as a universal barcode [16]. This approach circumvents the need for PCR, which can be problematic for preserved specimens with degraded DNA and also provides a method for less ambiguous reference databases for taxonomic identification [17]. Regardless of where the standard for DNA barcode technology is headed, barcode sequence databases will benefit from a growing number of sequences generated for known species.
