Efficient COI barcoding using high throughput single-end 400 bp sequencing
Chentao Yang, Yuxuan Zheng, Shangjin Tan, Guanliang Meng, Wei Rao, Cai‐qing Yang, David G. Bourne, Paul A. O’Brien, Junqiang Xu, Sha Liao, Ao Chen, Xiaowei Chen, Xinrui Jia, Ai‐bing Zhang, Shanlin Liu
Abstract
BACKGROUND: Over the last decade, the rapid development of high-throughput sequencing platforms has accelerated species description and assisted morphological classification through DNA barcoding. However, the current high-throughput DNA barcoding methods cannot obtain full-length barcode sequences due to read length limitations (e.g. a maximum read length of 300 bp for the Illumina's MiSeq system), or are hindered by a relatively high cost or low sequencing output (e.g. a maximum number of eight million reads per cell for the PacBio's SEQUEL II system). RESULTS: Pooled cytochrome c oxidase subunit I (COI) barcodes from individual specimens were sequenced on the MGISEQ-2000 platform using the single-end 400 bp (SE400) module. We present a bioinformatic pipeline, HIFI-SE, that takes reads generated from the 5' and 3' ends of the COI barcode region and assembles them into full-length barcodes. HIFI-SE is written in Python and includes four function modules of filter, assign, assembly and taxonomy. We applied the HIFI-SE to a set of 845 samples (30 marine invertebrates, 815 insects) and delivered a total of 747 fully assembled COI barcodes as well as 70 Wolbachia and fungi symbionts. Compared to their corresponding Sanger sequences (72 sequences available), nearly all samples (71/72) were correctly and accurately assembled, including 46 samples that had a similarity score of 100% and 25 of ca. 99%. CONCLUSIONS: The HIFI-SE pipeline represents an efficient way to produce standard full-length barcodes, while the reasonable cost and high sensitivity of our method can contribute considerably more DNA barcodes under the same budget. Our method thereby advances DNA-based species identification from diverse ecosystems and increases the number of relevant applications.