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    Laboratory Optimization Tweaks for Sanger Sequencing in a Resource-Limited Setting

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    Author
    Onwuamah, Chika K
    Okwuraiwe, Azuka P
    Ahmed, Rahaman A
    Sokei, Judith O
    Ponmak, Jamda
    Okoli, Leona C
    Kagurusi, Brian A
    Anejo-Okopi, Joseph
    Date
    2020-12-31
    Journal
    Journal of Biomolecular Techniques : JBT
    Publisher
    Association of Biomolecular Resource Facilities
    Type
    Article
    
    Metadata
    Show full item record
    See at
    https://doi.org/10.7171/jbt.20-3104-006
    Abstract
    Despite various challenges that hinder the implementation of high-tech molecular methods in resource-limited settings, we have been able to implement and achieve International Organization for Standardization 15189:2012 accreditation for genotypic HIV drug resistance testing in our facility. At the Center for Human Virology and Genomics, Nigerian Institute of Medical Research, Nigeria has recorded a high sequencing success rate and good quality sequence data. This was achieved by optimizing laboratory processes from 2008 to the current date. We have optimized sample preparation, RT-PCR, several post-PCR processes, and the cycle sequencing to improve the sensitivity of amplification even with limited plasma samples and low viral copy numbers. The optimized workflow maximizes output, minimizes reagent wastage, and achieves substantial cost savings without compromising the quality of the sequence data. Our performance at our last external quality assurance program is a testimonial to the efficiency of the workflow. For the 5-sample panel, each with 67–68 mutation points evaluated, we scored 100% for all 5 specimens. Our optimized laboratory workflow is thus documented to support laboratories and to help researchers achieve excellent results the first time and eliminate contamination while minimizing the wastage of costly sequencing reagents.
    Rights/Terms
    © Association of Biomolecular Resource Facilities.
    Keyword
    biomedical research
    HIV drug resistance
    laboratory workflow
    reverse transcriptase polymerase chain reaction
    Identifier to cite or link to this item
    http://hdl.handle.net/10713/14217
    ae974a485f413a2113503eed53cd6c53
    10.7171/jbt.20-3104-006
    Scopus Count
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