Milken Institute School of Public Health Poster Presentations (Marvin Center & Video)

Title

Specific Amplification of Esherichia coli Genomic Regions for Eventual Use in Targeted Amplicon Sequencing

Poster Number

51

Document Type

Poster

Status

Graduate Student - Masters

Abstract Category

Environmental and Occupational Health

Keywords

amplicon, E. coli

Publication Date

Spring 2018

Abstract

Background: Laboratory identification of Escherichia coli sequence types (STs) in a mixed sample remains a challenge, as commonly used genome sequencing regions do not provide enough resolution to characterize communities beyond the genus or species level. Multilocus sequence typing (MLST) is a robust method of strain typing for E. coli, but requires isolation of each sub-species. The importance of identifying particular E. coli sub-species is increasingly being recognized, and a need exists for a culture-independent laboratory assay capable of distinguishing the sub-species present within bacterial communities.

Methods: Candidate 500 base pair genomic loci were identified based on single nucleotide polymorphism (SNP) diversity and the number of clusters of genomes formed when each locus was evaluated against 5,000 E. coli genomes in silico. 20 initial loci were selected based on the feasibility of designing primers of an appropriate annealing temperature and with limited degeneracy at the 3’ end. These loci were screened using the polymerase chain reaction (PCR) and gel electrophoresis, to evaluate amplification of 12 non-E. coli Enterobacteriaceae species. To reduce non-specific amplification, a touch-down PCR protocol was developed and used for additional analysis of the 2 highest-performing loci (82752 and 143365). The DNA from 31 E. coli STs was quantified by qPCR, normalized to a starting input of 100,000 genomes, and serially diluted. Diluted E. coli DNA was then used to estimate limit of detection and confirm amplification of a range of diverse E. coli sub-species.

Results: On the initial tests of loci 82752 and 143365, visible bands were observed for only 2 non-E. coli organisms, but amplification of E. coli controls was sub-optimal. With lower annealing temperatures and lengthened cycles, all E. coli controls were amplified, but specificity to the E. coli genome was lost. Using touch down PCR, all E. coli controls were detected and non-specific amplification was minimized to 2-3 species/locus. Diversity panel testing indicated that both loci could be used to effectively amplify 31 E. coli STs. Preliminary limit of detection analysis showed visible bands for the 4 STs tested at a starting value of 1,000 genomes.

Conclusions: These results indicate the feasibility of selective amplification of a 500 bp region of the E. coli genome by touch down PCR, with minimal non-specific amplification of related organisms. Additional experimentation will focus on developing a targeted amplicon sequencing method specific to one of these E. coli loci.

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Specific Amplification of Esherichia coli Genomic Regions for Eventual Use in Targeted Amplicon Sequencing

Background: Laboratory identification of Escherichia coli sequence types (STs) in a mixed sample remains a challenge, as commonly used genome sequencing regions do not provide enough resolution to characterize communities beyond the genus or species level. Multilocus sequence typing (MLST) is a robust method of strain typing for E. coli, but requires isolation of each sub-species. The importance of identifying particular E. coli sub-species is increasingly being recognized, and a need exists for a culture-independent laboratory assay capable of distinguishing the sub-species present within bacterial communities.

Methods: Candidate 500 base pair genomic loci were identified based on single nucleotide polymorphism (SNP) diversity and the number of clusters of genomes formed when each locus was evaluated against 5,000 E. coli genomes in silico. 20 initial loci were selected based on the feasibility of designing primers of an appropriate annealing temperature and with limited degeneracy at the 3’ end. These loci were screened using the polymerase chain reaction (PCR) and gel electrophoresis, to evaluate amplification of 12 non-E. coli Enterobacteriaceae species. To reduce non-specific amplification, a touch-down PCR protocol was developed and used for additional analysis of the 2 highest-performing loci (82752 and 143365). The DNA from 31 E. coli STs was quantified by qPCR, normalized to a starting input of 100,000 genomes, and serially diluted. Diluted E. coli DNA was then used to estimate limit of detection and confirm amplification of a range of diverse E. coli sub-species.

Results: On the initial tests of loci 82752 and 143365, visible bands were observed for only 2 non-E. coli organisms, but amplification of E. coli controls was sub-optimal. With lower annealing temperatures and lengthened cycles, all E. coli controls were amplified, but specificity to the E. coli genome was lost. Using touch down PCR, all E. coli controls were detected and non-specific amplification was minimized to 2-3 species/locus. Diversity panel testing indicated that both loci could be used to effectively amplify 31 E. coli STs. Preliminary limit of detection analysis showed visible bands for the 4 STs tested at a starting value of 1,000 genomes.

Conclusions: These results indicate the feasibility of selective amplification of a 500 bp region of the E. coli genome by touch down PCR, with minimal non-specific amplification of related organisms. Additional experimentation will focus on developing a targeted amplicon sequencing method specific to one of these E. coli loci.