Browsing by Author "Ilahi, Wafa"
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- Metabarcoding identification of botanical origin of bee-collected pollen samples: comparison of ITS2 reference database performance before and after enrichment with sequences of bee plant speciesPublication . Ilahi, Wafa; Pinto, M. Alice; Quaresma, Andreia; Selmi, BoulbebaSequence analysis of complex DNA samples is an important approach to monitoring species distribution in biodiversity and population studies. Although many traditional methods can be used to identify bee plant resources (e.g., direct observations, microscopy), high-throughput sequencing technologies have transformed our ability to explore more complex plant communities without necessity of experts and in a time cost effective-manner, in comparison with the classical techniques. Recently, metabarcoding, which combines DNA barcoding with high-throughput sequencing, of bee pollen has been used to successfully identify which plants bees are foraging on. This is achieved most commonly by sequencing short, conserved marker genes amplified with universal PCR primers. The ITS region has already been proposed and mainly used as universal barcode marker for plants. ITS sequences are often compared against an annotated reference sequence database to identify the likely taxonomic origin of each sequence with as much specificity as possible. So, accurate and specific taxonomic information is an important step in pollen metabarcoding analysis. The aim of this study was to assess performance of the ITS2 reference database before and after enrichment with sequences of bee plant species collected from across Europe. A total of 100 plant samples were collected in six countries and were identified morphologically at the species level. The DNA of these plant samples was extracted and then Sanger-sequenced for the ITS2 region. The sequences were curated and blasted against ITS2 sequences in GenBank to obtain taxonomic identification and assess similarity with the morphological identification. Next, a reference database of ITS2 sequences was generated using the 100 sequences. The newly-developed sequences were added to a comprehensive ITS2 reference database publicly available and the performance of the two databases (with and without the 100 sequences generated herein) was compared on a set of 108 mixed pollen samples that were identified by metabarcoding and classical palynology. The analyses of the data showed a positive correlation (r>0.7; P<0.05) between the two methods at both family (most countries) and genus levels (one third of countries), even though metabarcoding identified 12 families and 26 genera and palynology 13 families and 22 plant genera. Classification of mixed pollen samples, using the ITS2 database before enrichment with the new sequences developed herein, retrieved (i) 76 and 67 families before and after removing taxa that are not present in Europe, respectively, and (ii) 69 families after enrichment. At the genus level, before database enrichment, there were 230 and 214 genera identified before and after removing taxa that are not present in Europe, respectively, and 216 genera after enrichment. Results showed a positive correlation (r>0.9; P<0.001) between the different databases, but variation in the number of families and genera was noticed, which indicates an increase in the resolution and accuracy of classification. This study thus offers improvements in the ITS2 metabarcoding with more available sequences in the reference dataset providing heightened sensitivity and resolution.