Pyrosequencing for mini-barcoding of fresh and old museum specimens

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 difficu...

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Bibliographic Details
Published in:PloS one 2011-07, Vol.6 (7), p.e21252-e21252
Main Authors: Shokralla, Shadi, Zhou, Xin, Janzen, Daniel H, Hallwachs, Winnie, Landry, Jean-François, Jacobus, Luke M, Hajibabaei, Mehrdad
Format: Article
Language:English
Subjects:
Animals
Automation
Bar codes
Base Sequence
Biodiversity
Biology
Cost analysis
Cryptic species
Cytochrome
Cytochrome-c oxidase
Deoxyribonucleic acid
DNA
DNA barcoding
DNA Barcoding, Taxonomic - methods
DNA fragmentation
DNA sequencing
Electron Transport Complex IV - genetics
Fragmentation
Fragments
Gene sequencing
Genomes
Identification
Instrumentation
Lepidoptera
Lepidoptera - enzymology
Lepidoptera - genetics
Library collections
Marine biology
Museums
Oxidases
Phylogeny
Quality
Sepsis
Taxonomy
Technology
Temperature
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0021252