Restriction enzyme digestion of host DNA enhances universal detection of parasitic pathogens in blood via targeted amplicon deep sequencing

Abstract

Background: Targeted amplicon deep sequencing (TADS) of the 16S rRNA gene is commonly used to explore and characterize bacterial microbiomes. Meanwhile, attempts to apply TADS to the detection and characterization of entire parasitic communities have been hampered since conserved regions of many conserved parasite genes, such as the 18S rRNA gene, are also conserved in their eukaryotic hosts. As a result, targeted amplification of 18S rRNA from clinical samples using universal primers frequently results in competitive priming and preferential amplification of host DNA. Here, we describe a novel method that employs a single pair of universal primers to capture all blood-borne parasites while reducing host 18S rRNA template and enhancing the amplification of parasite 18S rRNA for TADS. This was achieved using restriction enzymes to digest the 18S rRNA gene at cut sites present only in the host sequence prior to PCR amplification.

Results: This method was validated against 16 species of blood-borne helminths and protozoa. Enzyme digestion prior to PCR enrichment and Illumina amplicon deep sequencing led to a substantial reduction in human reads and a corresponding 5- to 10-fold increase in parasite reads relative to undigested samples. This method allowed for discrimination of all common parasitic agents found in human blood, even in cases of multi-parasite infection, and markedly reduced the limit of detection in digested versus undigested samples.

Conclusions: The results herein provide a novel methodology for the reduction of host DNA prior to TADS and establish the validity of a next-generation sequencing-based platform for universal parasite detection.

Keywords: Amplicon sequencing; Blood microbiota; Molecular parasitology; Parasite biodiversity.

Fig. 1

Reduction of host DNA by restriction enzyme digestion enhances PCR amplification of parasite DNA. DNA extraction from parasite-infected whole blood yields a DNA sample containing high amounts of host DNA (blue) and low amounts of parasite DNA (bright red). a Performing conventional PCR on this sample, using universal primers, amplifies primarily host DNA (blue), and yields sequencing reads almost entirely belonging to the host. b In contrast, restriction enzyme digestion of host DNA prior to PCR alters the ratio of host to parasite DNA in the initial sample, allowing for selective amplification of parasite DNA (bright red) and resulting in an increase in the relative number of parasite amplicons post-PCR and an increase in the sensitivity of parasite detection via NGS

Fig. 2

Digestion of host DNA increases the sensitivity of parasite detection in parasite-positive human blood samples. (a) Restriction enzyme digestion yields a marked reduction in human 18S rRNA reads per thousand (left panel, greyscale diamonds) and a 5- to 10-fold increase in parasite reads per thousand (right panel, colored circles) in digested relative to undigested samples (n = 3 biological replicates, mean ± SD, samples were normalized according to the reads per thousand for reads derived from human host and parasite separately, with the central dotted line reflective of a zero fold change, which marks the undigested samples before treatment with restriction enzymes). No statistical difference was found for size selection (i.e., > 2 kb vs. < 2 kb) (two-way ANOVA, p = 0.0631). (b) Proportional composition of human DNA dilutions in undigested (ud) and digested (d) samples demonstrates an average 2-fold reduction in human DNA and a 5-fold increase in parasite reads post-digestion (black bars = C. felis, dark grey bars = H. sapiens, light grey bars = P. falciparum, concentration of 3D7 DNA includes P. falciparum and H. sapiens DNA from 3D7 cultures which contain human blood products, two-way ANOVA with Sidak’s multiple comparisons posttest, p < 0.0001, n = 3, mean ± SD)

Fig. 3

Enzyme digestion enhances sensitivity of detection for mixed parasite infections. Restriction enzyme digestion of mixed parasite infections in human blood yields a clear reduction in human 18S rRNA reads per thousand (left panel, greyscale diamonds) and a 2- to 15-fold increase in parasite reads per thousand (right panel, colored circles). Samples were normalized according to the reads per thousand for reads derived from human host and parasite separately, with the central dotted line reflective of a zero fold change, which marks the undigested samples before treatment with restriction

Fig. 4

Enzyme digestion markedly lowers assay limit of detection. (a) Reads per thousand for undigested (gray) and digested (black) 10-fold serial dilutions of P. knowlesi in whole human blood (n = 4, mean ± SD). (b) Log-transformation of reads per thousand from serially diluted samples suggests a limit of detection of 163 parasites per microliter for undigested samples (gray, r² = 0.9852) and 15 parasites per microliter for digested samples (black, r² = 0.9533) (n = 4 biological replicates, mean ± SD). (c) After deeper analysis, reads per thousand for undigested (gray) and digested (black) serial dilutions between 61 parasites per microliter and 0.72 parasites per microliter demonstrate a limit of detection of 40 to 60 parasites per microliter for undigested samples and 7 to 29 parasites per microliter for digested samples (two-way ANOVA with Sidak’s multiple comparisons posttest, **** p < 0.0001, *** p < 0.001, ** p < 0.005 n = 3, mean ± SD)