Abstract
DNA barcoding is an effective approach for species identification and for discovery of new and/or cryptic species. Sanger sequencing technology is the method of choice for obtaining standard 650 bp cytochrome c oxidase subunit I (COI) barcodes. However, DNA degradation/fragmentation makes it difficult to obtain a full-length barcode from old specimens. Mini-barcodes of 130 bp from the standard barcode region have been shown to be effective for accurate identification in many animal groups and may be readily obtained from museum samples. Here we demonstrate the application of an alternative sequencing technology, the four-enzymes single-specimen pyrosequencing, in rapid, cost-effective mini-barcode analysis. We were able to generate sequences of up to 100 bp from mini-barcode fragments of COI in 135 fresh and 50 old Lepidoptera specimens (ranging from 53–97 year-old). The sequences obtained using pyrosequencing were of high quality and we were able to robustly match all the tested pyro-sequenced samples to their respective Sanger-sequenced standard barcode sequences, where available. Simplicity of the protocol and instrumentation coupled with higher speed and lower cost per sequence than Sanger sequencing makes this approach potentially useful in efforts to link standard barcode sequences from unidentified specimens to known museum specimens with only short DNA fragments.
Highlights
DNA sequences have become a major source of information for understanding biodiversity
Long-term preservation of biological samples may cause DNA shearing and DNA inter-strand crosslinking, which result in DNA degradation
DNA shearing is the break-down of DNA into small fragments, which might be introduced by poor storage conditions such as exposure to UV radiation, high temperature, pH, and salinity [3]
Summary
DNA sequences have become a major source of information for understanding biodiversity. A mini-barcoding approach, which focuses the analysis on shorter DNA fragments, has been shown to be effective in gaining DNA sequence information from old museum samples and a 130 bp fragment from the 59 end of the full-length DNA barcode region has shown to be effective in distinguishing up to 91% of animal species in a broad taxonomic range [4,5]. This same fragment has even shown promise for DNA analysis in benthic insect collections treated with formalin [6]
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