Quasispecies Analyses of the HIV-1 Near-full-length Genome with Illumina MiSeq
Human immunodeficiency virus type-1 (HIV-1) exhibits high between-host genetic diversity and within-host heterogeneity, recognized as quasispecies. Because HIV-1 quasispecies fluctuate in terms of multiple factors, such as antiretroviral exposure and host immunity, analyzing the HIV-1 genome is crit...
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2015-11-01
|
| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fmicb.2015.01258/full |
| _version_ | 1818700669660430336 |
|---|---|
| author | Hirotaka eOde Masakazu eMatsuda Kazuhiro eMatsuoka Atsuko eHachiya Junko eHattori Yumiko eKito Yoshiyuki eYokomaku Yasumasa eIwatani Yasumasa eIwatani Wataru eSugiura Wataru eSugiura |
| author_facet | Hirotaka eOde Masakazu eMatsuda Kazuhiro eMatsuoka Atsuko eHachiya Junko eHattori Yumiko eKito Yoshiyuki eYokomaku Yasumasa eIwatani Yasumasa eIwatani Wataru eSugiura Wataru eSugiura |
| author_sort | Hirotaka eOde |
| collection | DOAJ |
| description | Human immunodeficiency virus type-1 (HIV-1) exhibits high between-host genetic diversity and within-host heterogeneity, recognized as quasispecies. Because HIV-1 quasispecies fluctuate in terms of multiple factors, such as antiretroviral exposure and host immunity, analyzing the HIV-1 genome is critical for selecting effective antiretroviral therapy and understanding within-host viral coevolution mechanisms. Here, to obtain HIV-1 genome sequence information that includes minority variants, we sought to develop a method for evaluating quasispecies throughout the HIV-1 near-full-length genome using the Illumina MiSeq benchtop deep sequencer. To ensure the reliability of minority mutation detection, we applied an analysis method of sequence read mapping onto a consensus sequence derived from de novo assembly followed by iterative mapping and subsequent unique error correction. Deep sequencing analyses of a HIV-1 clone showed that the analysis method reduced erroneous base prevalence below 1% in each sequence position and discarded only <1% of all collected nucleotides, maximizing the usage of the collected genome sequences. Further, we designed primer sets to amplify the HIV-1 near-full-length genome from clinical plasma samples. Deep sequencing of 92 samples in combination with the primer sets and our analysis method provided sufficient coverage to identify >1%-frequency sequences throughout the genome. When we evaluated sequences of pol genes from 18 treatment-naïve patients’ samples, the deep sequencing results were in agreement with Sanger sequencing and identified numerous additional minority mutations. The results suggest that our deep sequencing method would be suitable for identifying within-host viral population dynamics throughout the genome. |
| first_indexed | 2024-12-17T15:08:37Z |
| format | Article |
| id | doaj.art-c76f72ca11a8448ba38711f004696bcd |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-17T15:08:37Z |
| publishDate | 2015-11-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-c76f72ca11a8448ba38711f004696bcd2022-12-21T21:43:44ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2015-11-01610.3389/fmicb.2015.01258168705Quasispecies Analyses of the HIV-1 Near-full-length Genome with Illumina MiSeqHirotaka eOde0Masakazu eMatsuda1Kazuhiro eMatsuoka2Atsuko eHachiya3Junko eHattori4Yumiko eKito5Yoshiyuki eYokomaku6Yasumasa eIwatani7Yasumasa eIwatani8Wataru eSugiura9Wataru eSugiura10National Hospital Organization Nagoya Medical CenterNational Hospital Organization Nagoya Medical CenterNational Hospital Organization Nagoya Medical CenterNational Hospital Organization Nagoya Medical CenterNational Hospital Organization Nagoya Medical CenterNational Hospital Organization Nagoya Medical CenterNational Hospital Organization Nagoya Medical CenterNational Hospital Organization Nagoya Medical CenterGraduate School of Medicine, Nagoya UniversityNational Hospital Organization Nagoya Medical CenterGraduate School of Medicine, Nagoya UniversityHuman immunodeficiency virus type-1 (HIV-1) exhibits high between-host genetic diversity and within-host heterogeneity, recognized as quasispecies. Because HIV-1 quasispecies fluctuate in terms of multiple factors, such as antiretroviral exposure and host immunity, analyzing the HIV-1 genome is critical for selecting effective antiretroviral therapy and understanding within-host viral coevolution mechanisms. Here, to obtain HIV-1 genome sequence information that includes minority variants, we sought to develop a method for evaluating quasispecies throughout the HIV-1 near-full-length genome using the Illumina MiSeq benchtop deep sequencer. To ensure the reliability of minority mutation detection, we applied an analysis method of sequence read mapping onto a consensus sequence derived from de novo assembly followed by iterative mapping and subsequent unique error correction. Deep sequencing analyses of a HIV-1 clone showed that the analysis method reduced erroneous base prevalence below 1% in each sequence position and discarded only <1% of all collected nucleotides, maximizing the usage of the collected genome sequences. Further, we designed primer sets to amplify the HIV-1 near-full-length genome from clinical plasma samples. Deep sequencing of 92 samples in combination with the primer sets and our analysis method provided sufficient coverage to identify >1%-frequency sequences throughout the genome. When we evaluated sequences of pol genes from 18 treatment-naïve patients’ samples, the deep sequencing results were in agreement with Sanger sequencing and identified numerous additional minority mutations. The results suggest that our deep sequencing method would be suitable for identifying within-host viral population dynamics throughout the genome.http://journal.frontiersin.org/Journal/10.3389/fmicb.2015.01258/fullDrug ResistanceHIV-1Deep sequencingerror correctionQuasispeciesconsensus sequence estimation |
| spellingShingle | Hirotaka eOde Masakazu eMatsuda Kazuhiro eMatsuoka Atsuko eHachiya Junko eHattori Yumiko eKito Yoshiyuki eYokomaku Yasumasa eIwatani Yasumasa eIwatani Wataru eSugiura Wataru eSugiura Quasispecies Analyses of the HIV-1 Near-full-length Genome with Illumina MiSeq Frontiers in Microbiology Drug Resistance HIV-1 Deep sequencing error correction Quasispecies consensus sequence estimation |
| title | Quasispecies Analyses of the HIV-1 Near-full-length Genome with Illumina MiSeq |
| title_full | Quasispecies Analyses of the HIV-1 Near-full-length Genome with Illumina MiSeq |
| title_fullStr | Quasispecies Analyses of the HIV-1 Near-full-length Genome with Illumina MiSeq |
| title_full_unstemmed | Quasispecies Analyses of the HIV-1 Near-full-length Genome with Illumina MiSeq |
| title_short | Quasispecies Analyses of the HIV-1 Near-full-length Genome with Illumina MiSeq |
| title_sort | quasispecies analyses of the hiv 1 near full length genome with illumina miseq |
| topic | Drug Resistance HIV-1 Deep sequencing error correction Quasispecies consensus sequence estimation |
| url | http://journal.frontiersin.org/Journal/10.3389/fmicb.2015.01258/full |
| work_keys_str_mv | AT hirotakaeode quasispeciesanalysesofthehiv1nearfulllengthgenomewithilluminamiseq AT masakazuematsuda quasispeciesanalysesofthehiv1nearfulllengthgenomewithilluminamiseq AT kazuhiroematsuoka quasispeciesanalysesofthehiv1nearfulllengthgenomewithilluminamiseq AT atsukoehachiya quasispeciesanalysesofthehiv1nearfulllengthgenomewithilluminamiseq AT junkoehattori quasispeciesanalysesofthehiv1nearfulllengthgenomewithilluminamiseq AT yumikoekito quasispeciesanalysesofthehiv1nearfulllengthgenomewithilluminamiseq AT yoshiyukieyokomaku quasispeciesanalysesofthehiv1nearfulllengthgenomewithilluminamiseq AT yasumasaeiwatani quasispeciesanalysesofthehiv1nearfulllengthgenomewithilluminamiseq AT yasumasaeiwatani quasispeciesanalysesofthehiv1nearfulllengthgenomewithilluminamiseq AT wataruesugiura quasispeciesanalysesofthehiv1nearfulllengthgenomewithilluminamiseq AT wataruesugiura quasispeciesanalysesofthehiv1nearfulllengthgenomewithilluminamiseq |