GeneSeq is a highly accurate genotypic HIV drug resistance test. GeneSeq detects the mutations associated with Nucleoside Reverse Transcriptase Inhibitors (NRTI), Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTI), and Protease Inhibitors (PI) resistance within the HIV genome. These results are analyzed through Monogram Biosciences' proprietary algorithms. These algorithms incorporate information from Monogram Biosciences' own database of more than 100,000 HIV samples with paired genotype and phenotype information. This sensitive and accurate test gives the healthcare providers a prediction of a patient's HIV resistance profile based on the genotype.

Performing the test

Sample preparation

DNA copies of the patient's HIV protease and reverse transcriptase genes are amplified from the viral RNA by RT-PCR (reverse transcription-polymerase chain reaction). The process has been optimized and validated to ensure reliable amplification samples with a viral load of 500 copies or more. The DNA copies are inserted into a resistance test vectors as populations of sequences that represent the diversity of the viral quasi-species present in the patient sample.

Nucleic acid sequencing

Nucleic acid sequencing for GeneSeq is based upon the dideoxynucleotide chain termination method of DNA sequencing (dideoxy sequencing)

1. Purified DNA is combined with specific primers, deoxy-nucleotides, dye-terminator dideoxy nucleotides, and a thermostable polymerase, and the resultant reaction mixtures are incubated in a thermal cycler.
2. The cycle-sequencing reaction produces a series of fluorescence-tagged DNA fragments that can be separated into discrete sizes by capillary electrophoresis in order to determine the order of nucleotides on the original HIV DNA.

Analyzing the data

Sequence interpretations (interpretation algorithm)

1. The DNA nucleotide sequence is translated into an amino acid sequence, and then compared to the sequence of a drug-sensitive ("wild-type") reference virus.
2. Changes in amino acid sequence (mutations) from the wild-type reference virus are identified and reported.
3. Based upon the mutations detected, an interpretation algorithm based on published literature, international expert recommendations, and in-house analysis of over 100,000 paired genotypes and phenotypes, is used to predict drugs to which the patient's virus would be expected to exhibit reduced susceptibility.