Browsing by Subject "high-throughput sequencing"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Effort Versus Reward: Preparing samples for fungal community characterization in high-throughput sequencing surveys of soils
    (2015-03-25) Song, Zewei; Schlatter, Dan; Kennedy, Peter; Kinkel, Linda; Kistler, H.Corby; Nguyen, Nhu; Bates, Scott; songx208@umn.edu; Song, Zewei
    Next generation fungal amplicon sequencing is being used with increasing frequency to study fungal diversity in various ecosystems; however, the influence of sample preparation on the characterization of fungal community is poorly understood. We investigated the effects of four procedural modifications to library preparation for high-throughput sequencing (HTS). The following treatments were considered: 1) the amount of soil used in DNA extraction, 2) the inclusion of additional steps (freeze/thaw cycles, sonication, or hot water bath incubation) in the extraction procedure, 3) the amount of DNA template used in polymerase chain reaction (PCR), and 4) the effect of sample pooling, either physically or computationally. Soils from two different ecosystems in Minnesota, USA, one prairie and one forest site, were used to assess the generality of our results. The first three treatments did not significantly influence observed fungal operational taxonomic unit (OTU) richness or community structure at either site. Physical pooling captured more OTU richness compared to individual samples, but total OTU richness at each site was highest when individual samples were computationally combined. We conclude that standard extraction kit protocols are well optimized for fungal HTS surveys, but because sample pooling can significantly influence OTU richness estimates, it is important to carefully consider the study aims when planning sampling procedures.
  • Thumbnail Image
    Item
    Immunological selection as a driver of porcine reproductive and respiratory syndrome virus (PRRSV) evolution
    (2016-07) Wang, Xiong
    Porcine reproductive and respiratory syndrome (PRRS) is still one of the most devastating swine infectious diseases worldwide since its initial outbreak in the late 1980s. Its etiologic agent, PRRS virus (PRRSV), is a single strand RNA virus that belongs to the order of Nidovirales, family Arteriviridae and genus Arterivirus. PRRSV is small, viral partial size is about 53nm including a RNA genome with the size of ~ 15kb. PRRSV is highly host restricted to porcine monocyte cells. Currently in the field, biosecurity and passive immunization are the major solutions to reducing the impact of PRRSV endemic. Yet two major factors, the rapidly evolution of PRRSV and the incomplete and highly variable cross-protection induced by passive vaccination, heavily contribute to the penetration of PRRSV to swine herds and result in the emergence and re-emergence of virulent PRRSV. Similar to other RNA virus like human immunodeficiency virus 1 (HIV-1), hepatitis C virus (HCV) and foot-and-mouth disease virus (FMDV), the lack of error prone mechanism during viral replication leads to the production of tremendous mutations in PRRSV progeny. The selection pressure of porcine intrinsic, innate and adaptive immunity on PRRSV population helps shape and drive PRRSV mutation direction. Together, they drive PRRSV’s rapid evolution. Yet there is a limitation in systemic understanding how genetic variation is generated and what selection forces drive PRRSV evolution. The overall objective of this dissertation was to characterize PRRSV evolution in intra-population and field level, as well as exploring the driving forces hidden in the porcine monocyte cells by utilizing high- throughput sequencing and bioinformatics. The findings herein built up an optimized standard protocol to assemble PRRSV whole genome from high-throughput sequencing yields, which can be broadly adapted to other highly-mutated RNA virus. PRRSV infectious clones, similar to field isolates, exist as quasispecies, its population diversity was decreasing under consistent selection pressure of permissive cell intrinsic selection yet retaining significant diversified progeny. In this thesis, an emerging virulent PRRSV in vaccinated herds was identified as a recently evolved member of virulent lineage instead of new virulent strain via built comprehensive and standard analysis pipeline. IFNs and IFN-induced ARFs are highly induced in PAMs after PRRSV inoculation; potential causative key pathways were identified in the PAM age-dependent susceptibility difference scenario. Putting together, all the findings and results provided an improved systematic insight of PRRSV evolution and host innate response, which is a vital immunological selection driver. Ultimately, a better understanding of PRRSV evolution and its driver will lead to a more effective disease prevention, control and elimination.