Comparison of a high-resolution melting assay to next-generation sequencing for analysis of HIV diversity.
Next-generation sequencing (NGS) has recently been used for analysis of HIV diversity, but this method is labor-intensive, costly, and requires complex protocols for data analysis. We compared diversity measures obtained using NGS data to those obtained using a diversity assay based on high-resolution melting (HRM) of DNA duplexes. The HRM diversity assay provides a single numeric score that reflects the level of diversity in the region analyzed. HIV gag and env from individuals in Rakai, Uganda, were analyzed in a previous study using NGS (n = 220 samples from 110 individuals). Three sequence-based diversity measures were calculated from the NGS sequence data (percent diversity, percent complexity, and Shannon entropy). The amplicon pools used for NGS were analyzed with the HRM diversity assay. HRM scores were significantly associated with sequence-based measures of HIV diversity for both gag and env (P < 0.001 for all measures). The level of diversity measured by the HRM diversity assay and NGS increased over time in both regions analyzed (P < 0.001 for all measures except for percent complexity in gag), and similar amounts of diversification were observed with both methods (P < 0.001 for all measures except for percent complexity in gag). Diversity measures obtained using the HRM diversity assay were significantly associated with those from NGS, and similar increases in diversity over time were detected by both methods. The HRM diversity assay is faster and less expensive than NGS, facilitating rapid analysis of large studies of HIV diversity and evolution.
Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Matthew M
MM
Cousins
San-San
SS
Ou
Maria J
MJ
Wawer
Supriya
S
Munshaw
David
D
Swan
Craig A
CA
Magaret
Caroline E
CE
Mullis
David
D
Serwadda
Stephen F
SF
Porcella
Ronald H
RH
Gray
Thomas C
TC
Quinn
Deborah
D
Donnell
Susan H
SH
Eshleman
Andrew D
AD
Redd
D000328:Adult; D004279:DNA, Viral / Q000235:genetics*; D014644:Genetic Variation; D006678:HIV / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D015658:HIV Infections / Q000821:virology*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008967:Molecular Biology / Q000379:methods; D009691:Nucleic Acid Denaturation; D044366:Transition Temperature; D014454:Uganda; D014773:Virology / Q000379:methods; D054299:env Gene Products, Human Immunodeficiency Virus / Q000235:genetics; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics
D004279:DNA, Viral; D054299:env Gene Products, Human Immunodeficiency Virus; D054301:gag Gene Products, Human Immunodeficiency Virus