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Gigascience. 2017 Dec 1;6(12):1-8. doi: 10.1093/gigascience/gix104.

Filling reference gaps via assembling DNA barcodes using high-throughput sequencing-moving toward barcoding the world.

Liu S1,2,3, Yang C2, Zhou C1,4, Zhou X1,5.

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Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Plant Protection, China Agricultural University, Beijing 100193, People's Republic of China.
BGI-Shenzhen, Shenzhen, 518083, China.
Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, 1350, Copenhagen, Denmark.
Key Laboratory of Bio-Resources and Eco-Environment, Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610065, China.
National Engineering Research Center for Fruit and Vegetable Processing, China Agricultural University, Beijing 100193, People's Republic of China.


Over the past decade, biodiversity researchers have dedicated tremendous efforts to constructing DNA reference barcodes for rapid species registration and identification. Although analytical cost for standard DNA barcoding has been significantly reduced since early 2000, further dramatic reduction in barcoding costs is unlikely because Sanger sequencing is approaching its limits in throughput and chemistry cost. Constraints in barcoding cost not only led to unbalanced barcoding efforts around the globe, but also prevented high-throughput sequencing (HTS)-based taxonomic identification from applying binomial species names, which provide crucial linkages to biological knowledge. We developed an Illumina-based pipeline, HIFI-Barcode, to produce full-length Cytochrome c oxidase subunit I (COI) barcodes from pooled polymerase chain reaction amplicons generated by individual specimens. The new pipeline generated accurate barcode sequences that were comparable to Sanger standards, even for different haplotypes of the same species that were only a few nucleotides different from each other. Additionally, the new pipeline was much more sensitive in recovering amplicons at low quantity. The HIFI-Barcode pipeline successfully recovered barcodes from more than 78% of the polymerase chain reactions that didn't show clear bands on the electrophoresis gel. Moreover, sequencing results based on the single molecular sequencing platform Pacbio confirmed the accuracy of the HIFI-Barcode results. Altogether, the new pipeline can provide an improved solution to produce full-length reference barcodes at about one-tenth of the current cost, enabling construction of comprehensive barcode libraries for local fauna, leading to a feasible direction for DNA barcoding global biomes.


Biodiveristy; COI; DNA Barcode; High-throughput sequencing; meta-barcoding

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