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- Brodin, Johanna, et al.
(författare)
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PCR-Induced Transitions Are the Major Source of Error in Cleaned Ultra-Deep Pyrosequencing Data
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:7
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Tidskriftsartikel (refereegranskat)abstract
- Background: Ultra-deep pyrosequencing (UDPS) is used to identify rare sequence variants. The sequence depth is influenced by several factors including the error frequency of PCR and UDPS. This study investigated the characteristics and source of errors in raw and cleaned UDPS data. Results: UDPS of a 167-nucleotide fragment of the HIV-1 SG3Denv plasmid was performed on the Roche/454 platform. The plasmid was diluted to one copy, PCR amplified and subjected to bidirectional UDPS on three occasions. The dataset consisted of 47,693 UDPS reads. Raw UDPS data had an average error frequency of 0.30% per nucleotide site. Most errors were insertions and deletions in homopolymeric regions. We used a cleaning strategy that removed almost all indel errors, but had little effect on substitution errors, which reduced the error frequency to 0.056% per nucleotide. In cleaned data the error frequency was similar in homopolymeric and non-homopolymeric regions, but varied considerably across sites. These site-specific error frequencies were moderately, but still significantly, correlated between runs (r = 0.15-0.65) and between forward and reverse sequencing directions within runs (r = 0.33-0.65). Furthermore, transition errors were 48-times more common than transversion errors (0.052% vs. 0.001%; p<0.0001). Collectively the results indicate that a considerable proportion of the sequencing errors that remained after data cleaning were generated during the PCR that preceded UDPS. Conclusions: A majority of the sequencing errors that remained after data cleaning were introduced by PCR prior to sequencing, which means that they will be independent of platform used for next-generation sequencing. The transition vs. transversion error bias in cleaned UDPS data will influence the detection limits of rare mutations and sequence variants.
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| 2. |
- Hedskog, Charlotte, et al.
(författare)
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Dynamics of HIV-1 Quasispecies during Antiviral Treatment Dissected Using Ultra-Deep Pyrosequencing
- 2010
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 5:7, s. e11345-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Ultra-deep pyrosequencing (UDPS) allows identification of rare HIV-1 variants and minority drug resistance mutations, which are not detectable by standard sequencing. Principal Findings: Here, UDPS was used to analyze the dynamics of HIV-1 genetic variation in reverse transcriptase (RT) (amino acids 180-220) in six individuals consecutively sampled before, during and after failing 3TC and AZT containing antiretroviral treatment. Optimized UDPS protocols and bioinformatic software were developed to generate, clean and analyze the data. The data cleaning strategy reduced the error rate of UDPS to an average of 0.05%, which is lower than previously reported. Consequently, the cut-off for detection of resistance mutations was very low. A median of 16,016 (range 2,406-35,401) sequence reads were obtained per sample, which allowed detection and quantification of minority resistance mutations at amino acid position 181, 184, 188, 190, 210, 215 and 219 in RT. In four of five pre-treatment samples low levels (0.07-0.09%) of the M184I mutation were observed. Other resistance mutations, except T215A and T215I were below the detection limit. During treatment failure, M184V replaced M184I and dominated the population in combination with T215Y, while wild-type variants were rarely detected. Resistant virus disappeared rapidly after treatment interruption and was undetectable as early as after 3 months. In most patients, drug resistant variants were replaced by wild-type variants identical to those present before treatment, suggesting rebound from latent reservoirs. Conclusions: With this highly sensitive UDPS protocol preexisting drug resistance was infrequently observed; only M184I, T215A and T215I were detected at very low levels. Similarly, drug resistant variants in plasma quickly decreased to undetectable levels after treatment interruption. The study gives important insights into the dynamics of the HIV-1 quasispecies and is of relevance for future research and clinical use of the UDPS technology.
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| 3. |
- Hedskog, Charlotte
(författare)
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Ultra-deep characterization of viral quasispecies in HIV infection
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- HIV-1 has the ability to rapidly diversify and adapt to changes in its environment, such as evading the host immune response, altering cell tropism, and developing resistance to antiretroviral drugs. Minority HIV-1 variants have been shown to be of clinical significance, especially those with non-nucleoside reverse transcriptase inhibitor (NNRTI) drug resistance mutations or determinants of CXCR4 phenotype (X4-virus). In this thesis a next generation sequencing technology, ultra-deep pyrosequencing (UDPS), has been used to dissect HIV-1 quasispecies in infected patients to study the evolution of drug resistance and cell tropism. The depth of UDPS depends on the number of viral templates that can be successfully extracted and amplified from a plasma sample, the error rate of PCR and UDPS, and the efficiency of cleaning the UDPS data from such errors. For this reason, we developed an experimental design that allows high recovery of HIV-1 templates and an efficient data cleaning strategy. Our data cleaning strategy reduced the UDPS error rate approximately 10-fold. We carefully evaluated the performance of our UDPS protocol and found that the repeatability of detection of major as well as minor variants in patient plasma samples was good. This indicated that the experimental noise introduced during RNA extraction, cDNA synthesis, PCR and UDPS was low. However, for rare variants in vitro PCR recombination and effects of sequence direction need to be considered. Finally, the design of primers for PCR amplification is of special importance during UDPS, since we observed that primer-related selective amplification can skew the frequency estimates of genetic variants. We investigated the levels of pre-existing drug resistance mutations in plasma samples from five treatment-naive patients. In four of five patients we found low levels of pre-existing drug resistance mutations at two positions (M184I, T215A/I), whereas other mutations (M184V, Y181C, Y188C and T215Y/F) were not detected. During treatment failure and treatment interruption, we found almost complete replacement of wild-type and drug-resistant variants, respectively. This implies that the proportion of minority variants with drug resistance in patients with previous treatment failure or transmitted drug resistance can be too low to be detectible even with highly sensitive UDPS. In another study, the HIV-1 populations from three patients with HIV-1 populations that switched coreceptor use were investigated longitudinally. UDPS analysis showed that the X4-virus that emerged after coreceptor switch was not detected during primary HIV-1 infection (PHI) and that the X4 population most probably evolved from the CCR5-using population during the course of infection rather than was transmitted as minor variants. Moreover, one to three major variants were found during PHI, supporting that infection usually is established with one or just a few viral particles. The frequency and type of errors that occurred during UDPS were investigated. The errors that remained after data cleaning were significantly more often transitions than transversions, which indicates that a substantial proportion of errors were introduced during PCR rather than UDPS itself. This affects the limits of detection of minority mutations since UDPS analyses of HIV-1 are preceded by a PCR step. To further reduce the UDPS error rate we developed a new, improved methodology, based on re-sequencing of molecularly tagged template molecules. Preliminary results showed that this method has the potential to increase the sensitivity of UDPS analyses 1000-fold and thus is close to error-free. Taken together, this thesis adds knowledge on the use of UDPS to gain new insights in HIV evolution and resistance and is relevant for the possible future clinical use of this technology.
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