OTUs and ASVs Produce Comparable Taxonomic and Diversity from Shrimp Microbiota 16S Profiles Using Tailored Abundance Filters.

Rodrigo García-López,Rogerio R Sotelo-Mundo,Alonso A Lopez-Zavala,Adrian Ochoa-Leyva,Fernanda Cornejo-Granados,Andrés Cota-Huízar,Bruno Gómez-Gil

doi:10.3390/genes12040564

Rodrigo García-López, Rogerio R Sotelo-Mundo + Show 5 more

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https://doi.org/10.3390/genes12040564

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Abstract

The interplay between shrimp immune system, its environment, and microbiota contributes to the organism’s homeostasis and optimal production. The metagenomic composition is typically studied using 16S rDNA profiling by clustering amplicon sequences into operational taxonomic units (OTUs) and, more recently, amplicon sequence variants (ASVs). Establish the compatibility of the taxonomy, α, and β diversity described by both methods is necessary to compare past and future shrimp microbiota studies. Here, we used identical sequences to survey the V3 16S hypervariable-region using 97% and 99% OTUs and ASVs to assess the hepatopancreas and intestine microbiota of L. vannamei from two ponds under standardized rearing conditions. We found that applying filters to retain clusters >0.1% of the total abundance per sample enabled a consistent taxonomy comparison while preserving >94% of the total reads. The three sets turned comparable at the family level, whereas the 97% identity OTU set produced divergent genus and species profiles. Interestingly, the detection of organ and pond variations was robust to the clustering method’s choice, producing comparable α and β-diversity profiles. For comparisons on shrimp microbiota between past and future studies, we strongly recommend that ASVs be compared at the family level to 97% identity OTUs or use 99% identity OTUs, both using tailored frequency filters.

Highlights

Crustacean production is one of the fastest-growing economic activities to embrace aquaculture as its primary source for commercial produce [1], with over 9.38 million tons of specimens produced worldwide as of 2018, accounting for 22% of the international aquatic species market
Illumina adapters were removed, obtaining a total of 957,415 reads (39,892.29 ± 8592.20), which were used for amplicon sequence variants (ASVs) and operational taxonomic units (OTUs) taxonomic profiling (Supplementary Table S3.1)
V3 sequences were subjected to different quality filters and sequence-joining procedures to create three types of sequence clusters, two from OTUs at 97 and 99% identity and one for ASVs, see Experimental Procedures (Figure 1C, Supplementary Figure S2.1)

Summary

Introduction

Crustacean production is one of the fastest-growing economic activities to embrace aquaculture as its primary source for commercial produce [1], with over 9.38 million tons of specimens produced worldwide as of 2018, accounting for 22% of the international aquatic species market. Since the Pacific whiteleg shrimp, L. vannamei, is the most commonly cultured shrimp species worldwide [2], several studies have explored its genetics and, more recently, the impact of bacteria in its digestive tract both under wild-type and standardized environmental condition in farms [3,4,5,6,7,8]. A previous study determined that the hypervariable region V3 is a costeffective alternative to determine the microbiota diversity in the shrimp hepatopancreas and the intestine [12]. Amplicons spanning both the V3 and V4 regions bore a higher taxonomic resolution and diversity, the V3 region showed optimal family-level resolution and better performance than V4 at the genus level using current sequencing technologies [12]

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