P25.03
Background: Assays for identification of HIV drug resistance (DR) mutations are becoming expensive and laborious due to growth of quantity of HIV-infected people and number of drugs. Next generation sequencing (NGS) has a significant advantage over conventional population sequencing which is used in routine diagnostic. Implementation of NGS in diagnostic practice will greatly reduce the complexity and the cost of DR testing. Our aim was to develop the molecular method for deep sequencing of all viral genes and compare the results with conventional population sequencing.
Methods: The blood plasma from HIV-positive person who has been infected in 1990 and changed 5 schemes of therapy at the sampling date was used as a clinical material. SuperScript III RT-PCR System was applied for RT-PCR and in-house reagents for the second round of amplification. Totally there were amplified more than 9000 nucleotides with 5 overlapping fragments. These fragments were sequenced using ABI 3500 and MiSeq in parallel. Data obtained by the population sequencing were analyzed by BioEdit and DEONA software. Analysis of NGS data was done by the in-house algorithm.
Results: Following results were obtained from the NGS data: total capacity −1.018.551.807 nucleotides, 7.748.528 reads; total length of the consensus sequence −9.581 nucleotides (98.58% of HXB2); maximum depth coverage −283.055 reads; average −80.128 reads; depth less than 1000 reads −189 nucleotides (1,97%).
The concordance between the consensus sequences of NGS and the population sequence (total length - 6363 nucleotides) was 99,25%. Thorough analysis revealed that only 48 positions were different and there were found complete mismatches only in 10 positions.
NGS consensus contained all DR mutations which were found in population consensus.
Conclusions: We developed the molecular method which allows getting near full HIV genome by amplification of overlapping fragments and following NGS. Comparison with population sequencing revealed the high degree of concordance.
