Presentation Title

Sequencing and Hybrid Assembly of Antibiotic Resistant Bacteria from an Undergraduate Microbiology Course

Faculty Mentor

Paul Orwin

Start Date

23-11-2019 8:00 AM

End Date

23-11-2019 8:45 AM

Location

71

Session

poster 1

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Here we present the fully assembled genomes of three bacterial isolates using a hybrid assembly approach. Students in an upper division medical microbiology course isolated antibiotic resistant organisms from various environmental sources. Three isolates were identified as being from the genera Aeromonas, Comamonas, and Acinetobacter and were chosen for further investigation. All three of these genera are gram-negative proteobacteria known to include pathogenic species. The three microorganisms were isolated and confirmed to have resistance to several antibiotics. An internal region of the 16s rRNA gene was amplified by Polymerase Chain Reaction and sequenced using traditional Sanger sequencing. High concentration and high molecular weight preparations of genomic DNA were created using a modified alkaline lysis protocol. These preparations were sequenced using the MinION system (Oxford Nanopore). Additional genomic DNA was prepared using the Wizard Genomic DNA Preparation Kit (Promega), and short read sequencing was performed using the iSEQ 100 system (Illumina). The Illumina iSeq yielded accurate but short DNA sequences and the Oxford Nanopore MinION yielded long but error prone DNA sequences. We used various tools on Galaxy (usegalaxy.org), an open source web-based bioinformatic database, to trim and check the quality of the DNA sequences. To assemble the genomes, we used Unicycler. Unicycler is a tool found on Galaxy that combines data from the two sequencing technologies we used in this research to create a hybrid assembly. The genomes were compared to already assembled genomes of our three genera to confirm whether or not our species have been identified previously. Antibiotic resistance was already observed so we annotated the genomes using Prokka to search for antibiotic resistance and other virulence genes, as well as the presence of mobile elements to facilitate transfer of these genes.

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Nov 23rd, 8:00 AM Nov 23rd, 8:45 AM

Sequencing and Hybrid Assembly of Antibiotic Resistant Bacteria from an Undergraduate Microbiology Course

71

Here we present the fully assembled genomes of three bacterial isolates using a hybrid assembly approach. Students in an upper division medical microbiology course isolated antibiotic resistant organisms from various environmental sources. Three isolates were identified as being from the genera Aeromonas, Comamonas, and Acinetobacter and were chosen for further investigation. All three of these genera are gram-negative proteobacteria known to include pathogenic species. The three microorganisms were isolated and confirmed to have resistance to several antibiotics. An internal region of the 16s rRNA gene was amplified by Polymerase Chain Reaction and sequenced using traditional Sanger sequencing. High concentration and high molecular weight preparations of genomic DNA were created using a modified alkaline lysis protocol. These preparations were sequenced using the MinION system (Oxford Nanopore). Additional genomic DNA was prepared using the Wizard Genomic DNA Preparation Kit (Promega), and short read sequencing was performed using the iSEQ 100 system (Illumina). The Illumina iSeq yielded accurate but short DNA sequences and the Oxford Nanopore MinION yielded long but error prone DNA sequences. We used various tools on Galaxy (usegalaxy.org), an open source web-based bioinformatic database, to trim and check the quality of the DNA sequences. To assemble the genomes, we used Unicycler. Unicycler is a tool found on Galaxy that combines data from the two sequencing technologies we used in this research to create a hybrid assembly. The genomes were compared to already assembled genomes of our three genera to confirm whether or not our species have been identified previously. Antibiotic resistance was already observed so we annotated the genomes using Prokka to search for antibiotic resistance and other virulence genes, as well as the presence of mobile elements to facilitate transfer of these genes.