Optimization of 18 S rRNA metabarcoding for the simultaneous diagnosis of intestinal parasites

Abstract

Recent advancements in next-generation sequencing (NGS) technologies have created new opportunities for comprehensive screening of multiple parasite species. In this study, we cloned the 18 S rDNA V9 region of 11 species of intestinal parasites into plasmids. Equal amounts and concentrations of these 11 plasmids were pooled, and amplicon NGS targeting the 18 S rDNA V9 region was performed using the Illumina iSeq 100 platform. A total of 434,849 reads were identified, and all 11 parasite species were detected, although the number of output reads for each parasite varied. The read count ratio, in descending order, was as follows: Clonorchis sinensis, 17.2%; Entamoeba histolytica, 16.7%; Dibothriocephalus latus, 14.4%; Trichuris trichiura, 10.8%; Fasciola hepatica, 8.7%; Necator americanus, 8.5%; Paragonimus westermani, 8.5%; Taenia saginata, 7.1%; Giardia intestinalis, 5.0%; Ascaris lumbricoides, 1.7%; and Enterobius vermicularis, 0.9%. We found that the DNA secondary structures showed a negative association with the number of output reads. Additionally, variations in the amplicon PCR annealing temperature affected the relative abundance of output reads for each parasite. These findings can be applied to improve parasite detection methodologies and ultimately enhance efforts to control and prevent intestinal parasitic infections.

Keywords: 18S rDNA; Helminths; Intestinal parasites; Metabarcoding; Next generation sequencing; Protozoa.

Fig. 1

Flow chart outlining the sample preparation and amplicon sequencing process.

Fig. 2

Relative abundances of the sequence reads in 11 intestinal parasite species: Ascaris lumbricoides, Clonorchis sinensis, Dibothriocephalus latus, Entamoeba histolytica, Enterobius vermicularis, Fasciola hepatica, Giardia intestinalis, Necator americanus, Paragonimus westermani, Taenia saginata, and Trichuris trichiura.

Fig. 3

Secondary structure of the 18 S rDNA V9 region and GC pair numbers. (a) Trichuris trichiura, (b) Fasciola hepatica, (c) Clonorchis sinensis, (d) Dibothriocephalus latus, (e) Enterobius vermicularis, (f) Necator americanus, (g) Giardia intestinalis, (h) Entamoeba histolytica, (i) Paragonimus westermani, (j) Ascaris lumbricoides, (k) Taenia saginata.

Fig. 4

Bar plot showing the relative abundances of the plasmids of 11 species. Plasmid concentration at (a) 20 ng/µl and (b) 2 ng/µl. Bottom, no restriction enzyme treatment was performed; middle, the 11 plasmids were pooled first and then treated with the restriction enzyme, Nco1; top, each plasmid was first treated with the restriction enzyme (Nco1) and then pooled.

Fig. 5

Effect of annealing temperature on the relative abundance of sequence reads in 11 intestinal parasites.