Evaluating the accuracy and sensitivity of detecting minority HIV-1 populations by Illumina next-generation sequencing

• Published in: Journal of virological methods Vol. 261; pp. 40 - 45
• Main Authors: Kireev, D.E., Lopatukhin, A.E., Murzakova, A.V., Pimkina, E.V., Speranskaya, A.S., Neverov, A.D., Fedonin, G.G., Fantin, Yu. S., Shipulin, G.A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.11.2018
• Subjects: Accuracy; Deep sequencing; Genetic Variation; High-Throughput Nucleotide Sequencing - methods; HIV Infections - virology; HIV-1; HIV-1 - classification; HIV-1 - isolation & purification; Humans; Minor viral population; Sensitivity; Sensitivity and Specificity; HIV-1; Deep sequencing; Accuracy; Sensitivity; Minor viral population
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2018.08.001

•The type of nucleic acid and reverse transcription step do not affect the sequencing accuracy.•Accuracy and sensitivity critically depend on the coverage and threshold of sensitivity to minor nucleotides.•Sensitivity to minor variants of less than 1% leads to a significant decline in sequencing accuracy. The accuracy and sensitivity of deep sequencing were assessed using viral standards (pNL4-3 and pLAI.2) of both DNA and RNA. The sequencing accuracy did not depend on the type of nucleic acid, but critically depended on the number of reads and threshold of sensitivity to minor viral populations. With coverage of more than 236 reads, the accuracy of viral RNA sequencing was equal to or exceeded 99.9%, with a sensitivity threshold to minor nucleotides of 20%. When the sensitivity threshold was below 1%, reduced accuracy dynamics were clearly visible even when the coverage was massive (more than 9.000 reads). It was found that the floating sensitivity threshold allowed the sequencing accuracy to be maintained at an acceptable level in cases of low coverage (less than 1.500–2.000) of reads. These results indicate the quality that can be expected with a specific number of reads and sensitivity threshold. Deep sequencing is a very powerful tool that can significantly improve the value of study results, but despite its superior performance, it should be used with caution regarding its sensitivity to minor populations below 1%.