Details:

Alternative Title:
Parasitol Res
Personal Author:
Gondard, Mathilde ; Lane, Meredith ; Barratt, Joel ; Talundzic, Eldin ; Qvarnstrom, Yvonne
Gondard, Mathilde ; Lane, Meredith ; Barratt, Joel ; Talundzic, Eldin ; Qvarnstrom, Yvonne Less -
Description:
We recently described a targeted amplicon deep sequencing (TADS) strategy that utilizes a nested PCR targeting the 18S rDNA gene of blood-borne parasites. The assay facilitates selective digestion of host DNA by targeting enzyme restriction sites present in vertebrates but absent in parasites. This enriching of parasite-derived amplicon drastically reduces the proportion of host-derived reads during sequencing and results in the sensitive detection of several clinically important blood parasites including Plasmodium spp., Babesia spp., kinetoplastids, and filarial nematodes. Despite these promising results, high costs and the laborious nature of metagenomics sequencing are prohibitive to the routine use of this assay in most laboratories. We describe and evaluate a new metagenomic approach that utilizes a set of primers modified from our original assay that incorporates Illumina barcodes and adapters during the PCR steps. This modification makes amplicons immediately compatible with sequencing on the Illumina MiSeq platform, removing the need for a separate library preparation, which is expensive and time-consuming. We compared this modified assay to our previous nested TADS assay in terms of preparation speed, limit of detection (LOD), and cost. Our modifications reduced assay turnaround times from 7 to 5 days. The cost decreased from approximately $40 per sample to $11 per sample. The modified assay displayed comparable performance in the detection and differentiation of human-infecting Plasmodium spp., Babesia spp., kinetoplastids, and filarial nematodes in clinical samples. The LOD of this modified approach was determined for malaria parasites and remained similar to that previously reported for our earlier assay (0.58 Plasmodium falciparum parasites/µL of blood). These modifications markedly reduced costs and turnaround times, making the assay more amenable to routine diagnostic applications.
Keywords:
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Amplicon Sequencing Animals Babesia Cost-Benefit Analysis DNA, Ribosomal High-Throughput Nucleotide Sequencing Humans Library Preparation MiSeq Sequencing Molecular Diagnosis Molecular Parasitology Parasite Detection Parasites Plasmodium Plasmodium Falciparum
Source:
Parasitol Res. 122(12):3243-3256
Pubmed ID:
37940706
Pubmed Central ID:
PMC10772880
Document Type:
Journal Article
Funding:
CC999999/ImCDC/Intramural CDC HHSUnited States/
Collection(s):
CDC Public Access
Main Document Checksum:
[+]
urn:sha256:ce6ade3e3014846696f5e691795c3f547efda8a92ac686bbebd2f345c13dcf30
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