64 Standardizing a method for sequencing the entire HIV genome by massive semiconductor sequencing Margarita Matias-Florentino1, Santiago Ávila- Ríos1, Aarón Lecanda- Sánchez1 and Gustavo Reyes- Terán1. 1Center for Research in Infectious Diseases, National Institute of Respiratory Diseases, México City, México. Next-generation sequencing (NGS) technologies allow the generation of large volumes of data in a cost-effective manner. These advantages make it possible to sequence complete HIV genomes with a deep coverage, and allows for the detection of low frequency variants. However, this approach entails challenges in experimental design and bioinformatic analysis. We present preliminary results on a project aiming to characterize the genetic diversity of circulating HIV in the Mesoamerican region using whole genome sequences, and to determine the role of minority variants in HIV immune escape and antiretroviral (ARV) drug resistance. Peripheral blood samples were obtained from ARV treatment-naïve, HIV-infected patients from Mexico and Central America. Viral cDNA was produced from free plasma virus and then amplified using a nested PCR strategy covering the entire viral genome with three amplicons. Libraries for multiplex NGS runs were created and template for sequencing was produced. The enriched templates were sequenced using 316v2 chips in the Ion PGM platform. Approximately 13 patients were analyzed per run optimizing read yield without compromising coverage and depth. To assemble the viral genomes different strategies are being tested, HXB2 (K03455) and an HIV-B isolate (GQ372988) were used as references, using the aligners T-Map, SMALT and Bowtie2 with different parameters. The best alignment results were obtained with TMap and SMALT and were very similar for the two references, with 90% of the sequenced bases aligned and average coverage of 4,000X per patient. However, the coverage drastically dropped in the region between bases 6000 and 8000, corresponding to the env gene (800X), with a conflictive zone below 50X. In order to solve this problem a pipeline using T-map and a three steps reiterative alignment strategy is being developed.