From sequence data to patient result: a solution for HIV drug resistance genotyping with Exatype, end to end software for Pol-HIV-1 Sanger based Sequence analysis and patient HIV drug resistance result generation

Abstract

Introduction: With the rapid scale-up of antiretroviral therapy (ART) to treat HIV infection, there are ongoing concerns regarding probable emergence and transmission of HIV drug resistance (HIVDR) mutations. This scale-up has to lead to an increased need for routine HIVDR testing to inform the clinical decision on a regimen switch. Although the majority of wet laboratory processes are standardized, slow, labor-intensive data transfer and subjective manual sequence interpretation steps are still required to finalize and release patient results. We thus set out to validate the applicability of a software package to generate HIVDR patient results from raw sequence data independently.Methods: We assessed the performance characteristics of Hyrax Bioscience's Exatype (a sequence data to patient result, fully automated sequence analysis software, which consolidates RECall, MEGA X and the Stanford HIV database) against the standard method (RECall and Stanford database). Exatype is a web-based HIV Drug resistance bioinformatic pipeline available at sanger.exatype.com. To validate the exatype, we used a test set of 135 remnant HIV viral load samples at the National HIV Reference Laboratory (NHRL).Result: We analyzed, and successfully generated results of 126 sequences out of 135 specimens by both standard and Exatype software. Result production using Exatype required minimal hands-on time in comparison to the standard (6 computation-hours using the standard method versus 1.5 Exatype computation-hours). Concordance between the two systems was 99.8% for 311,227 bases compared. 99.7 % of the 0.2% discordant bases, were attributed to nucleotide mixtures as a result of the sequence editing in Recall. Both methods identified similar (99.1%) critical antiretroviral resistance-associated mutations resulting in a 99.2% concordance of resistance susceptibility interpretations. Base calling comparison between the two methods had Cohen's kappa (0.97 to 0.99) implying an almost perfect agreement with minimal base calling variation. On a predefined dataset, RECall editing displayed the highest probability to score mixtures accurately one vs. 0.71 and the lowest chance to inaccurately assign mixtures to pure nucleotides (0.002–0.0008). This advantage is attributable to the manual sequence editing in RECall.Conclusion: The reduction in hands-on time needed is a benefit when using the Exatype HIV DR sequence analysis platform and result generation tool. There is a minimal difference in base calling between Exatype and standard methods. Although the discrepancy has minimal impact on drug resistance interpretation, allowance of sequence editing in Exatype as RECall can significantly improve its performance.

Genomic epidemiology of SARS-CoV-2 during the first four waves in Mozambique

“…Raw reads from the Illumina COVIDSeq protocol were assembled using the Exatype NGS SARS-CoV-2 pipeline v1.6.1 (https://sars-cov-2. exatype.com/) [20]. Alternatively, samples sequenced from the Nanopore GridION were assembled with the Arctic-nCoV2019 SARS-CoV-2 assembly pipeline (https://artic.network/ ncov-2019/ncov2019-bioinformatics-sop.html).…”

“…For samples generated by RCB, raw reads were analyzed with the SARSCOVseq pipeline (https://github.com/ngs-fzb/SARSCOV2seq). Briefly, the reads were mapped with BWA [20,22] to the MN908947.3 reference and mappings were refined by INDEL realignment with GATK 3.8 [21,23]. Amplicon primer were trimmed after alignment and aberrant amplicon reads crossing expected amplicon boundaries were removed with iVar [22,24], also trimming for base quality 20 in a sliding window of 4 and removing unmapped and < = 30bp reads.…”

Need assessment for HIV drug resistance testing and landscape of current and future technologies in low- and middle-income countries

“…Starting from extracted HIV RNA, the kit includes modules for RT-PCR of the pol gene and sequencing on one of the Thermo Fisher sequencing platforms (Table 3). While the kit itself stops at the generation of raw sequence data, the company recommends basecalling with the Exatype [27] (Hyrax Biosciences) or ReCall [28] software to generate a consensus sequence, Lab-developed genotypic tests. Generally, tests performed in research laboratories do not have regulatory approval for use in individual patient management in North America or Europe.…”

Implementation of an interactive mobile application to pilot a rapid assay to detect HIV drug resistance mutations in Kenya

“…Due to the complexity of HIV drug resistance ( HIVDR ) tests used to guide treatment regimens, they are performed in centralized, highly-equipped laboratories [10–13]. In low-resource countries with high HIV prevalence like Kenya, few laboratories have the capacity to test for HIVDR [14]. For laboratories without access to sequencers, an oligonucleotide ligation assay ( OLA ) has been implemented [15] but onboarding OLA required extensive training due to its complexity.…”