Abstract
Metagenomic sequencing of patient samples is a very promising method for the diagnosis of human infections. Sequencing has the ability to capture all the DNA or RNA from pathogenic organisms in a human sample. However, complete and accurate characterization of the sequence, including identification of any pathogens, depends on the availability and quality of genomes for comparison. Thousands of genomes are now available, and as these numbers grow, the power of metagenomic sequencing for diagnosis should increase. However, recent studies have exposed the presence of contamination in published genomes, which when used for diagnosis increases the risk of falsely identifying the wrong pathogen. To address this problem, we have developed a bioinformatics system for eliminating contamination as well as low-complexity genomic sequences in the draft genomes of eukaryotic pathogens. We applied this software to identify and remove human, bacterial, archaeal, and viral sequences present in a comprehensive database of all sequenced eukaryotic pathogen genomes. We also removed low-complexity genomic sequences, another source of false positives. Using this pipeline, we have produced a database of “clean” eukaryotic pathogen genomes for use with bioinformatics classification and analysis tools. We demonstrate that when attempting to find eukaryotic pathogens in metagenomic samples, the new database provides better sensitivity than one using the original genomes while offering a dramatic reduction in false positives.
Highlights
Next-generation sequencing in pathogen discovery/diagnosisNext-generation sequencing (NGS) over the last few years has emerged as a valuable tool for human pathogen discovery and diagnosis
In 2013 Loman et al conducted a retrospective investigation into the 2011 German outbreak of Shiga-toxigenic Escherichia coli (STEC) [2]
In 2016, Salzberg et al tested the possibilities of detecting pathogens by sequencing brain or spinal cord biopsies from 10 patients presenting with neurologic symptoms with previously unidentified infections [5]
Summary
Next-generation sequencing (NGS) over the last few years has emerged as a valuable tool for human pathogen discovery and diagnosis. In 2014, Hasman et al analyzed 35 urine samples from patients with suspected urinary tract infections, confirming cultured bacterial infections using sequencing of isolated and cultured bacteria [3]. They successfully identified polymicrobial bacterial infections by directly sequencing the urine samples. NGS identified both bacterial and viral infections in selected patients, diagnoses that were confirmed by traditional immunologic techniques
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