The present study is aimed at implementing the morphological identification-free amplicon sequence variant (ASV) concept for describing meiofaunal species composition, while strongly indicating reasonable compatibility with the underlying species. A primer pair was constructed and demonstrated to PCR amplify a 470-490 bp 18S barcode from a variety of meiofaunal taxa, high throughput sequenced using the Illumina 300 x 2 bps platform. Sixteen 18S multi-species HTS assemblies were created from meiofaunal samples and merged to one assembly of similar to 2,150,000 reads. Five quality scores (q = 35, 30, 25, 20, 15) were implemented to filter five 18S barcode assemblies, which served as inputs for the DADA2 software, ending with five reference ASV libraries. Each of these libraries was clustered, applying 3% dissimilarity threshold, revealed an average number of 1.38 +/- 0.078 ASVs / cluster. Hence, demonstrating high level of ASV uniqueness. The libraries which were based on q <= 25 reached a near-asymptote number of ASVs which together with the low average number of ASVs / cluster, strongly indicated fair representation of the actual number of the underlying species. Hence, the q = 25 library was selected to be used as metabarcoding reference library. It contained 461 ASVs and 342-3% clusters with average number of 1.34 +/- 1.036 ASV / cluster and their BLASTN annotation elucidated a variety of expected meiofaunal taxa. The sixteen assemblies of sample-specific paired reads were mapped to this reference library and sample ASV profiles, namely the list of ASVs and their proportional copy numbers were created and clustered.

Amplicon sequence variant-based meiofaunal community composition revealed by DADA2 tool is compatible with species composition

Pallavicini, Alberto;
2022-01-01

Abstract

The present study is aimed at implementing the morphological identification-free amplicon sequence variant (ASV) concept for describing meiofaunal species composition, while strongly indicating reasonable compatibility with the underlying species. A primer pair was constructed and demonstrated to PCR amplify a 470-490 bp 18S barcode from a variety of meiofaunal taxa, high throughput sequenced using the Illumina 300 x 2 bps platform. Sixteen 18S multi-species HTS assemblies were created from meiofaunal samples and merged to one assembly of similar to 2,150,000 reads. Five quality scores (q = 35, 30, 25, 20, 15) were implemented to filter five 18S barcode assemblies, which served as inputs for the DADA2 software, ending with five reference ASV libraries. Each of these libraries was clustered, applying 3% dissimilarity threshold, revealed an average number of 1.38 +/- 0.078 ASVs / cluster. Hence, demonstrating high level of ASV uniqueness. The libraries which were based on q <= 25 reached a near-asymptote number of ASVs which together with the low average number of ASVs / cluster, strongly indicated fair representation of the actual number of the underlying species. Hence, the q = 25 library was selected to be used as metabarcoding reference library. It contained 461 ASVs and 342-3% clusters with average number of 1.34 +/- 1.036 ASV / cluster and their BLASTN annotation elucidated a variety of expected meiofaunal taxa. The sixteen assemblies of sample-specific paired reads were mapped to this reference library and sample ASV profiles, namely the list of ASVs and their proportional copy numbers were created and clustered.
https://www.sciencedirect.com/science/article/pii/S1874778722000587
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11368/3034400
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