Sequence Quality Analysis Tool (SQUAT) for HIV-1 Protease and Reverse Transcriptase.

Background: Access to antiretroviral therapy is increasing globally and drug resistance evolution is anticipated. Currently, Protease (PR) and reverse transcriptase (RT) sequence generation is increasing, including the use of in-house sequencing assays, and quality-assessment prior to sequence analysis is essential. Methods: We created a computational HIV PR/RT Sequence Quality Analysis Tool (SQUAT) that runs in the R statistical environment. Sequence quality thresholds are calculated from a large dataset (46,802 PR and 44,432 RT sequences) from the published literature (http://hivdb.Stanford.edu). Results: Nucleic acid sequences are read into SQUAT, identified, aligned and translated. Nucleic acid sequences are flagged if with >five 1-2-base insertions; >one 3-base insertion; >one deletion; >six PR or >18 RT ambiguous bases; >three consecutive PR or >four RT nucleic acid mutations; >zero stop codons; >three PR or >six RT ambiguous amino acids; >three consecutive PR or >four RT amino acid mutations, >zero unique amino acids; or <0.5% or >15% genetic distance from another submitted sequence. Thresholds are user-modifiable. A summary report details comments for troubleshooting of flagged sequences; histograms of pairwise genetic distances; neighbor joining phylogenetic trees; and aligned nucleic and amino acid sequences. Conclusions: SQUAT is a stand-alone, free, web-independent tool to ensure use of high-quality HIV PR/RT sequences in interpretation and reporting of drug resistance, while increasing awareness and expertise and facilitating troubleshooting of potentially problematic sequences.