Abstract
BackgroundHistorically, identification of causal agents of disease has relied heavily on the ability to culture the organism in the laboratory and/or the use of pathogen-specific antibodies or sequence-based probes. However, these methods can be limiting: Even highly sensitive PCR-based assays must be continually updated due to signature degradation as new target strains and near neighbors are sequenced. Thus, there has been a need for assays that do not suffer as greatly from these limitations and/or biases. Recent advances in library preparation technologies for Next-Generation Sequencing (NGS) are focusing on the use of targeted amplification and targeted enrichment/capture to ensure that the most highly discriminating regions of the genomes of known targets (organism-unique regions and/or regions containing functionally important genes or phylogenetically-discriminating SNPs) will be sequenced, regardless of the complex sample background.ResultsIn the present study, we have assessed the feasibility of targeted sequence enhancement via amplification to facilitate detection of a bacterial pathogen present in low copy numbers in a background of human genomic material. Our results indicate that the targeted amplification of signature regions can effectively identify pathogen genomic material present in as little as 10 copies per ml in a complex sample. Importantly, the correct species and strain calls could be made in amplified samples, while this was not possible in unamplified samples.ConclusionsThe results presented here demonstrate the efficacy of a targeted amplification approach to biothreat detection, using multiple highly-discriminative amplicons per biothreat organism that provide redundancy in case of variation in some primer regions. Importantly, strain level discrimination was possible at levels of 10 genome equivalents. Similar results could be obtained through use of panels focused on the identification of amplicons targeted for specific genes or SNPs instead of, or in addition to, those targeted for specific organisms (ongoing gene-targeting work to be reported later). Note that without some form of targeted enhancement, the enormous background present in complex clinical and environmental samples makes it highly unlikely that sufficient coverage of key pathogen(s) present in the sample will be achieved with current NGS technology to guarantee that the most highly discriminating regions will be sequenced.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1530-0) contains supplementary material, which is available to authorized users.
Highlights
Identification of causal agents of disease has relied heavily on the ability to culture the organism in the laboratory and/or the use of pathogen-specific antibodies or sequence-based probes
Comparison of fragment libraries versus AmpliSeqTM libraries In every case, the AmpliSeqTM libraries demonstrated a sizable increase in the number of pathogen-specific reads with a concomitant reduction in human reads as compared to fragment libraries prepared from the same samples (Fig. 1)
Owing to the design of the primers in the targeted amplification panel, all reads from those amplicons are guaranteed to be informative at the species-resolution level. (Since variations in targeted organisms could cause individual primers to fail, we designed multiple highly-discriminating amplicons to provide redundancy.) This contrasts to the unbiased sequencing, where any reads mapping to the pathogen come from random locations, many of which are common to multiple species at higher taxonomic levels of resolution
Summary
Identification of causal agents of disease has relied heavily on the ability to culture the organism in the laboratory and/or the use of pathogen-specific antibodies or sequence-based probes. These methods can be limiting: Even highly sensitive PCR-based assays must be continually updated due to signature degradation as new target strains and near neighbors are sequenced. Nucleic acid based methods of pathogen detection, such as PCR, robust and sensitive, are often subject to false positive and false negative readings. Strain variation at the nucleotide level can lead to signature erosion This is especially true in organisms with high mutation rates [4]. The number of primer pairs necessary to detect a large set of organisms or strains is often untenable or requires multiple rounds of primer design, complex bioinformatic software or both [6]
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