Tackling critical parameters in metazoan meta-barcoding experiments: a preliminary study based on coxI DNA barcode

PeerJ

article

Bachir Balech¹  Anna Sandionigi³  Caterina Manzari¹  Emiliano Trucchi⁴  Apollonia Tullo¹  Flavio Licciulli⁵  Giorgio Grillo⁵  Elisabetta Sbisà⁵  Stefano De Felici⁴  Cecilia Saccone⁷  Anna Maria D’Erchia⁷  Donatella Cesaroni⁴  Maurizio Casiraghi³  Saverio Vicario⁸ 

[1] Istituto di Biomembrane;Dipartimento di Biologia, Università degli studi di Bari ‘Aldo Moro’;Dipartimento di Biotecnologie e Bioscienze–Zooplantlab, Università degli studi di Milano Bicocca;Dipartimento di Biologia, Università di Roma Tor Vergata;Istituto di Tecnologie Biomediche–Consiglio Nazionale delle Ricerche;Istituto di Biologia Agroambientale e Forestale–Consiglio Nazionale delle Ricerche;Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari ‘Aldo Moro’;Istituto sull’Inquinamento Atmosferico–Consiglio Nazionale delle Ricerche

关键词: GC content;    DNA metabarcoding;    Amplification bias;    Taxonomic assignment;    coxI;    Biodiversity;    Carabids;    High Throughput Sequencing;   

DOI  :  10.7717/peerj.4845

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

Nowadays DNA meta-barcoding is a powerful instrument capable of quickly discovering the biodiversity of an environmental sample by integrating the DNA barcoding approach with High Throughput Sequencing technologies. It mainly consists of the parallel reading of informative genomic fragment/s able to discriminate living entities. Although this approach has been widely studied, it still needs optimization in some necessary steps requested in its advanced accomplishment. A fundamental element concerns the standardization of bioinformatic analyses pipelines. The aim of the present study was to underline a number of critical parameters of laboratory material preparation and taxonomic assignment pipelines in DNA meta-barcoding experiments using the cytochrome oxidase subunit-I (coxI) barcode region, known as a suitable molecular marker for animal species identification. We compared nine taxonomic assignment pipelines, including a custom in-house method, based on Hidden Markov Models. Moreover, we evaluated the potential influence of universal primers amplification bias in qPCR, as well as the correlation between GC content with taxonomic assignment results. The pipelines were tested on a community of known terrestrial invertebrates collected by pitfall traps from a chestnut forest in Italy. Although the present analysis was not exhaustive and needs additional investigation, our results suggest some potential improvements in laboratory material preparation and the introduction of additional parameters in taxonomic assignment pipelines. These include the correct setup of OTU clustering threshold, the calibration of GC content affecting sequencing quality and taxonomic classification, as well as the evaluation of PCR primers amplification bias on the final biodiversity pattern. Thus, careful attention and further validation/optimization of the above-mentioned variables would be required in a DNA meta-barcoding experimental routine.

【 授权许可】

CC BY