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Low-host double MDA workflow for uncultured ASFV positive blood and serum sample sequencing.

ABSTRACT: African swine fever (ASF) is a highly lethal and contagious disease caused by African swine fever virus (ASFV). Whole-genome sequencing of ASFV is necessary to study its mutation, recombination, and trace its transmission. Uncultured samples have a considerable amount of background DNA, which causes waste of sequencing throughput, storage space, and computing resources. Sequencing methods attempted for uncultured samples have various drawbacks. In this study, we improved C18 spacer MDA (Multiple Displacement Amplification)-combined host DNA exhaustion strategy to remove background DNA and fit NGS and TGS sequencing. Using this workflow, we successfully sequenced two uncultured ASFV positive samples. The results show that this method can significantly reduce the percentage of background DNA. We also developed software that can perform real-time base call and analyses in set intervals of ASFV TGS sequencing reads on a cloud server.

SUBMITTER: Zhang C

PROVIDER: S-EPMC9531595 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Low-host double MDA workflow for uncultured ASFV positive blood and serum sample sequencing.

Zhang Chengjun C Cheng Tangyu T Li Dongfan D Yu Xuexiang X Chen Fangzhou F He Qigai Q

Frontiers in veterinary science 20220920

African swine fever (ASF) is a highly lethal and contagious disease caused by African swine fever virus (ASFV). Whole-genome sequencing of ASFV is necessary to study its mutation, recombination, and trace its transmission. Uncultured samples have a considerable amount of background DNA, which causes waste of sequencing throughput, storage space, and computing resources. Sequencing methods attempted for uncultured samples have various drawbacks. In this study, we improved C18 spacer MDA (Multiple ...[more]

PMID: 36204298

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Project description:Sample pooling is a promising strategy to facilitate COVID-19 surveillance testing for a larger population in comparison to individual single testing due to resource and time constraints. Increased surveillance testing capacity will reduce the likelihood of outbreaks as the general population is returning to work, school, and other gatherings. We have analyzed the impact of three variables on the effectiveness of pooling test samples: swab type, workflow, and positive sample order. We investigated the performance of several commercially available swabs (Steripack polyester flocked, Puritan nylon flocked, Puritan foam) in comparison to a new injected molded design (Yukon). The bench-top performance of collection swab was conducted with a previously developed anterior nasal cavity tissue model, based on a silk-glycerol sponge to mimic soft tissue mechanics and saturated with a physiologically relevant synthetic nasal fluid spiked with heat-inactivated SARS-CoV-2. Overall, we demonstrated statistically significant differences in performance across the different swab types. A characterization of individual swab uptake (gravimetric analysis) and FITC microparticle release suggests that differences in absorbance and retention drive the observed differences in Ct of the pooled samples. We also proposed two distinct pooling workflows to encompass different community collection modes and analyzed the difference in resulting positive pools as an effect of workflow, swab type, and positive sample order. Overall, swab types with lower volume retention resulted in reduced false negative occurrence, also observed for collection workflows with limited incubation times. Concurrently, positive sample order did have a significant impact on pooling test outcome, particularly in the case of swab type with great volume retention. We demonstrated that the variables investigated here affect the results of pooled COVID-19 testing, and therefore should be considered while designing pooled surveillance testing.

Dataset Information

Low-host double MDA workflow for uncultured ASFV positive blood and serum sample sequencing.

Publications

Low-host double MDA workflow for uncultured ASFV positive blood and serum sample sequencing.

Similar Datasets

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