Deciphering HIV genetic variability and evolution by massive parallel pyrosequencing and bioinformatics

HIV-1 is a virus with a very variable genome and therefore has the ability to adapt to new environments which include escape from immune pressure and suboptimal antiretroviral treatment. Next-generation sequencing (NGS), especially ultra-deep pyrosequencing (UDPS), has enabled in-depth sequencing studies with a previously unattainable resolution. However, the technology is more error prone than traditional sequencing which makes it challenging to interpret UDPS results.

In this thesis we carried out comprehensive work to identify, characterize and reduce errors as well as investigate the UDPS performance (Papers II, III and IV). In Papers IV and V we used UDPS to study HIV-1 minority variants. Novel primer design software was developed in Paper I and a new method to tag molecules was developed and evaluated in Paper VI. The design of primers is of special importance in NGS to avoid selective amplification which may skew estimates of variant frequencies.

We developed a computer program, PrimerDesign, to meet the changed requirements for primer design. PrimerDesign is tailored to design primers from a multiple alignment and is suitable for all types of NGS that is preceded by amplification. The new Primer ID methodology has the potential to provide highly accurate deep sequencing. We identified three major challenges; a skewed resampling of Primer IDs, low recovery of templates and erroneous consensus sequences. Undetected this would lead to an underestimation in diversity of the quasispecies and cause a skewed and incorrect results. As many of our other findings, the methodology is not limited to HIV or virology.

The resolution of UDPS analysis is primarily determined by the number of input DNA templates, the error frequency of the method and the efficiency of data cleaning. In Papers II and IV we therefore optimized the pre-UDPS protocol and investigated the characteristics and sources of errors that occurred when UDPS was used to sequence a fragment of the HIV-1 pol gene. UDPS introduced indel errors located in homopolymeric regions that were removed by our in-house data cleaning software. The remaining errors were primarily substitution errors that were introduced in the PCR that preceded UDPS. Transitions were significantly more frequent than transversions, which will limit detection of minor variants and mutations in HIV-1 as well as other species. Further, we evaluated the quality and reproducibility of the UDPS technology in analysis of the same pol gene fragment. We concluded that the UDPS repeatability was good for both major and minor variants. In our experimental settings, in vitro recombination and sequencing directions posed a minor problem, but still needs to be considered especially for studies of minor viral variants and linkage between mutations.

Minority resistance mutations have been shown to impact the clinical outcome in treated patients. We examined the presence of pre-existing drug resistance mutations in treatment-naïve HIV-1 infected individuals and found very low levels of M184I, T215A and T215I, but no presence of M184V, Y181C, Y188C or T215Y/F. This indicates that the natural occurrence of these mutations is very low. When the same individuals experienced treatment failure or interruption, almost 100 % of the wild-type virus respective drug resistance variants were replaced. Other patients were followed from primary HIV infection (PHI) until their virus switched coreceptor use from CCR5 (R5) to CXCR4 (X4). We did not find any X4-using virus present as a minority population during PHI. The results indicate that the X4-using population most probably evolved in stepwise fashion from the R5-using populations in each of the three patients.

In conclusion, we have developed and used new NGS and bioinformatic methods to study HIV-1 genetic variation. We have shown that UDPS can be used to gain new insights in HIV evolution and to detect minority drug resistance mutations as well as minority variants.

List of scientific papers

I. Brodin J, Krishnamoorthy M, Athreya G, Fischer W, Hraber P, Gleasner C, Green L, Korber B, Leitner T. A multiple-alignment based primer design algorithm for genetically highly variable DNA targets. BMC Bioinformatics. 2013 Aug 21;14:255.
https://doi.org/10.1186/1471-2105-14-255

II. Brodin J, Mild M, Hedskog C, Sherwood E, Leitner T, Andersson B, Albert J. PCR-induced transitions are the major source of error in cleaned ultra-deep pyrosequencing data. PLoS One. 2013 Jul 23;8(7):e70388.
https://doi.org/10.1371/journal.pone.0070388

III. Hedskog C, Brodin J, Heddini A, Bratt G, Albert J, Mild M. Longitudinal ultradeep characterization of HIV type 1 R5 and X4 subpopulations in patients followed from primary infection to coreceptor switch. AIDS Res Hum Retroviruses. 2013 Sep;29(9):1237-44.
https://doi.org/10.1089/AID.2012.0349

IV. Mild M, Hedskog C, Jernberg J, Albert J. Performance of ultra-deep pyrosequencing in analysis of HIV-1 pol gene variation. PLoS One. 2011;6(7):e22741.
https://doi.org/10.1371/journal.pone.0022741

V. Hedskog C, Mild M, Jernberg J, Sherwood E, Bratt G, Leitner T, Lundeberg J, Andersson B, Albert J. Dynamics of HIV-1 quasispecies during antiviral treatment dissected using ultra-deep pyrosequencing. PLoS One. 2010 Jul 7;5(7):e11345.
https://doi.org/10.1371/journal.pone.0011345

VI. Brodin J, Hedskog C, Heddini A, Benard E, Neher R, Mild M, Albert J. Challenges with using Primer IDs to improve accuracy of next generation sequencing. [Manuscript]