Abstract
BackgroundTargeted 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.ResultsThis 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.ConclusionsThe 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.
Highlights
Targeted amplicon deep sequencing (TADS) of the 16S rRNA gene is commonly used to explore and characterize bacterial microbiomes
This system utilizes a minimum cutoff for positivity based on the average proportion of contaminating reads obtained per negative control specimen over multiple replicate analyses
Using this dual-criterion system, specimens were considered positive only if more than 20 reads mapped to the respective parasite reference sequence and if the number of parasite derived reads mapped to that reference sequence exceeded the shifting maximum cutoff value
Summary
Targeted amplicon deep sequencing (TADS) of the 16S rRNA gene is commonly used to explore and characterize bacterial microbiomes. Much of this work has depended on metagenomic and metatranscriptomic approaches, including whole genome shotgun sequencing of entire microbial communities [1,2,3] Such approaches are frequently applied to viral and Flaherty et al Microbiome (2018) 6:164 detecting C5-methylating eukaryotic pathogens [13,14,15]. A recent study sought to overcome this challenge by utilizing host DNA blocking primers in the assessment of parasite biodiversity in the feces of wild rats [5] This method was rarely able to achieve species-level identification, and application of the method to assess helminth biodiversity required worm isolation from fecal samples and amplification with class-specific primers [4]
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have