Towards Plant Species Identification in Complex Samples: A Bioinformatics Pipeline for the Identification of Novel Nuclear Barcode Candidates.

Alexandre Angers-Loustau,Patricia E Rischitor,Mauro Petrillo,Dafni M Kagkli,Maddalena Querci,Antonio Puertas Gallardo,Valentina Paracchini,Alex Patak,Joachim Kreysa

doi:10.1371/journal.pone.0147692

Abstract

Monitoring of the food chain to fight fraud and protect consumer health relies on the availability of methods to correctly identify the species present in samples, for which DNA barcoding is a promising candidate. The nuclear genome is a rich potential source of barcode targets, but has been relatively unexploited until now. Here, we show the development and use of a bioinformatics pipeline that processes available genome sequences to automatically screen large numbers of input candidates, identifies novel nuclear barcode targets and designs associated primer pairs, according to a specific set of requirements. We applied this pipeline to identify novel barcodes for plant species, a kingdom for which the currently available solutions are known to be insufficient. We tested one of the identified primer pairs and show its capability to correctly identify the plant species in simple and complex samples, validating the output of our approach.

Highlights

The development of analytical techniques for the correct analysis of plant species in complex samples is of paramount importance for the implementation of various legislations aiming at protecting the rights and the safety of the consumers
In order to be useful as a DNA barcode, a target needs to satisfy three crucial conditions: firstly, it should be short enough to allow efficient amplification and sequencing; secondly, it should be flanked by regions of high identity across species of interest, to allow the design of broad specificity primers; thirdly, the sequence of the amplified region itself should be sufficiently variable between species to allow assignment of sequenced amplicons to specific species of origin
Using the currently available whole plant genome sequences and common bioinformatics tools, the pipeline outputs the sequences of potential primers and associated amplicons that were computed to fulfil the described DNA barcode requirements

Summary

Introduction

The development of analytical techniques for the correct analysis of plant species in complex samples is of paramount importance for the implementation of various legislations aiming at protecting the rights and the safety of the consumers. Identification of Novel Plant Nuclear Barcode Candidates of individual assays for each of the taxons to be detected in a sample, requiring a priori hypotheses on the species present. For this reason, recent efforts were made to apply the concepts of DNA barcoding in this context [7][8]. In order to be useful as a DNA barcode, a target needs to satisfy three crucial conditions: firstly, it should be short enough to allow efficient amplification and sequencing; secondly, it should be flanked by regions of high identity across species of interest, to allow the design of broad specificity primers; thirdly, the sequence of the amplified region itself should be sufficiently variable between species to allow assignment of sequenced amplicons to specific species of origin

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Conclusion