A Preliminary Evaluation of the Performance of RNase H2-dependent PCR (rhAmpSeq) for Resistome Characterization in a Complex Sample
Open Access
- Author:
- Sapre, Anjali
- Area of Honors:
- Immunology and Infectious Disease
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Jasna Kovac, Thesis Supervisor
Pamela Hankey Giblin, Thesis Honors Advisor - Keywords:
- Antimicrobial Resistance
Genotyping
PCR
Antimicrobial Resistance Genes
Resistome Characterization
Foodborne Pathogen - Abstract:
- Genotyping is important in detecting antimicrobial resistance genes (ARGs) in a sample (i.e., resistome). Resistome characterization is technically challenging since ARGs constitute a small portion of the entire host (e.g., human, animal, or plant) genome. The current state-of-the art method for comprehensive resistome characterization is shotgun metagenomics, which requires sequencing at cost-prohibitive depth. Therefore, alternative, cost-effective methods for rapid resistome characterization are needed. Here, a novel RNase H2-dependent PCR (rhAmpSeq) method combined with amplicon sequencing was applied to a human fecal sample to assess the method’s limit of detection and primer amplification efficiency. To carry out preliminary assessment of the limit of detection and primer efficiency of the rhAmpSeq, a human fecal sample DNA was inoculated with decreasing concentrations of DNA extracted from a Shigella sonnei isolate, which contained unique ARGs that were not present in the fecal sample DNA. This allowed for assessment of the relationship between the counts of unique ARGs and the concentration of S. sonnei carrying the unique ARGs. Samples underwent rhAmp PCR 1 for target gene amplification, PCR 2 for Illumina barcoding, and Illumina sequencing using a MiSeq. The sequencing reads were mapped with AmrPlusPlus to ARGs present in the MEGARes 2.0 database. The relationship between number of reads mapping to unique genes and their concentration was assessed using a goodness-of-fit method. A goodness-of-fit analysis results indicated a negative correlation between the ARG counts and the dilution of S. sonnei DNA inoculated into a fecal sample DNA. For ARGs with counts <25, there was a poor correlation between number of ARGs and the dilution of S. sonnei, suggesting poor amplification efficiency. For ARGs with counts ≥25, there was a strong correlation between ARG count and the dilution of S. sonnei, suggesting good amplification efficiency. rhAmpSeq detected ARGs present in 0.001% of the total DNA. Additionally, 84.2% of the total unique ARGs present in the genome of S. sonnei only that were targeted by primers and were not present in the fecal sample were also detected via the rhAmpSeq method. These results indicate that rhAmpSeq has a good limit of detection and a potential to be developed as an alternative to costly shotgun metagenomics. The findings provide the foundation for further investigation and improvement of the method’s sensitivity and specificity for detecting individual ARGs.