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

Autor:	Onwuamah CK; Center for Human Virology and Genomics, Nigerian Institute of Medical Research, Yaba 101212, Lagos, Nigeria., Okwuraiwe AP; Center for Human Virology and Genomics, Nigerian Institute of Medical Research, Yaba 101212, Lagos, Nigeria., Ahmed RA; Center for Human Virology and Genomics, Nigerian Institute of Medical Research, Yaba 101212, Lagos, Nigeria., Sokei JO; Center for Human Virology and Genomics, Nigerian Institute of Medical Research, Yaba 101212, Lagos, Nigeria., Ponmak J; Center for Human Virology and Genomics, Nigerian Institute of Medical Research, Yaba 101212, Lagos, Nigeria., Okoli LC; Center for Human Virology and Genomics, Nigerian Institute of Medical Research, Yaba 101212, Lagos, Nigeria., Kagurusi BA; University of Maryland, Baltimore, Maryland, USA; and., Anejo-Okopi J; AIDS Prevention Initiative in Nigeria, Jos, University Teaching Hospital, Jos, Nigeria.
Jazyk:	angličtina
Zdroj:	Journal of biomolecular techniques : JBT [J Biomol Tech] 2020 Dec; Vol. 31 (4), pp. 157-164.
DOI:	10.7171/jbt.20-3104-006
Abstrakt:	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. (© Association of Biomolecular Resource Facilities.)
Databáze:	MEDLINE
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