Offline Next Generation Metagenomics Sequence Analysis Using MinION Detection Software (MINDS).

Field laboratories interested in using the MinION often need the internet to perform sample analysis. Thus, the lack of internet connectivity in resource-limited or remote locations renders downstream analysis problematic, resulting in a lack of sample identification in the field. Due to this dependency, field samples are generally transported back to the lab for analysis where internet availability for downstream analysis is available. These logistics problems and the time lost in sample characterization and identification, pose a significant problem for field scientists. To address this limitation, we have developed a stand-alone data analysis packet using open source tools developed by the Nanopore community that does not depend on internet availability. Like Oxford Nanopore Technologies' (ONT) cloud-based What's In My Pot (WIMP) software, we developed the offline MinION Detection Software (MINDS) based on the Centrifuge classification engine for rapid species identification. Several online bioinformatics applications have been developed surrounding ONT's framework for analysis of long reads. We have developed and evaluated an offline real time classification application pipeline using open source tools developed by the Nanopore community that does not depend on internet availability. Our application has been tested on ATCC's 20 strain even mix whole cell (ATCC MSA-2002) sample. Using the Rapid Sequencing Kit (SQK-RAD004), we were able to identify all 20 organisms at species level. The analysis was performed in 15 min using a Dell Precision 7720 laptop. Our offline downstream bioinformatics application provides a cost-effective option as well as quick turn-around time when analyzing samples in the field, thus enabling researchers to fully utilize ONT's MinION portability, ease-of-use, and identification capability in remote locations.

Regulation of capsule biosynthesis in serotype A strains of Pasteurella multocida.

The capsule of Pasteurella multocida serotype A strain ATCC 11039 is composed of hyaluronic acid and is an important virulence factor. Repeated subculturing of certain capsular serotype A strains results in dissociation from a capsulated to a noncapsulated phenotype with a concomitant loss of virulence. Although noncapsulated variants have been thought to arise as a result of mutation, the molecular mechanisms underlying this event are unknown. In this study, we demonstrate that restoration of the capsulated phenotype occurs in vivo subsequent to intraperitoneal inoculation of BALB/c mice with a noncapsulated variant. Moreover, reverse transcription polymerase chain reaction analysis revealed the capsule locus to be under transcriptional control. Cloning and sequencing of a 290-bp fragment within the promoter containing intergenic region of the capsule locus of 11039/iso revealed no significant alterations occurred subsequent to subculturing. These results demonstrate that serotype A P. multocida strain ATCC 11039 regulates capsule expression in response to an unidentified environmental factor(s), thereby providing insights into the molecular mechanisms underlying colonial dissociation.