Analysis of 16S rRNA amplicon sequencing options on the Roche/454 next-generation titanium sequencing platform.
BACKGROUND: 16S rRNA gene pyrosequencing approach has revolutionized studies in microbial ecology. While primer selection and short read length can affect the resulting microbial community profile, little is known about the influence of pyrosequencing methods on the sequencing throughput and the out...
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2011-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3179495?pdf=render |
| _version_ | 1818292830408278016 |
|---|---|
| author | Hideyuki Tamaki Chris L Wright Xiangzhen Li Qiaoyan Lin Chiachi Hwang Shiping Wang Jyothi Thimmapuram Yoichi Kamagata Wen-Tso Liu |
| author_facet | Hideyuki Tamaki Chris L Wright Xiangzhen Li Qiaoyan Lin Chiachi Hwang Shiping Wang Jyothi Thimmapuram Yoichi Kamagata Wen-Tso Liu |
| author_sort | Hideyuki Tamaki |
| collection | DOAJ |
| description | BACKGROUND: 16S rRNA gene pyrosequencing approach has revolutionized studies in microbial ecology. While primer selection and short read length can affect the resulting microbial community profile, little is known about the influence of pyrosequencing methods on the sequencing throughput and the outcome of microbial community analyses. The aim of this study is to compare differences in output, ease, and cost among three different amplicon pyrosequencing methods for the Roche/454 Titanium platform METHODOLOGY/PRINCIPAL FINDINGS: The following three pyrosequencing methods for 16S rRNA genes were selected in this study: Method-1 (standard method) is the recommended method for bi-directional sequencing using the LIB-A kit; Method-2 is a new option designed in this study for unidirectional sequencing with the LIB-A kit; and Method-3 uses the LIB-L kit for unidirectional sequencing. In our comparison among these three methods using 10 different environmental samples, Method-2 and Method-3 produced 1.5-1.6 times more useable reads than the standard method (Method-1), after quality-based trimming, and did not compromise the outcome of microbial community analyses. Specifically, Method-3 is the most cost-effective unidirectional amplicon sequencing method as it provided the most reads and required the least effort in consumables management. CONCLUSIONS: Our findings clearly demonstrated that alternative pyrosequencing methods for 16S rRNA genes could drastically affect sequencing output (e.g. number of reads before and after trimming) but have little effect on the outcomes of microbial community analysis. This finding is important for both researchers and sequencing facilities utilizing 16S rRNA gene pyrosequencing for microbial ecological studies. |
| first_indexed | 2024-12-13T03:06:12Z |
| format | Article |
| id | doaj.art-05022b4949924e92a87887b61818b933 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-13T03:06:12Z |
| publishDate | 2011-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-05022b4949924e92a87887b61818b9332022-12-22T00:01:43ZengPublic Library of Science (PLoS)PLoS ONE1932-62032011-01-0169e2526310.1371/journal.pone.0025263Analysis of 16S rRNA amplicon sequencing options on the Roche/454 next-generation titanium sequencing platform.Hideyuki TamakiChris L WrightXiangzhen LiQiaoyan LinChiachi HwangShiping WangJyothi ThimmapuramYoichi KamagataWen-Tso LiuBACKGROUND: 16S rRNA gene pyrosequencing approach has revolutionized studies in microbial ecology. While primer selection and short read length can affect the resulting microbial community profile, little is known about the influence of pyrosequencing methods on the sequencing throughput and the outcome of microbial community analyses. The aim of this study is to compare differences in output, ease, and cost among three different amplicon pyrosequencing methods for the Roche/454 Titanium platform METHODOLOGY/PRINCIPAL FINDINGS: The following three pyrosequencing methods for 16S rRNA genes were selected in this study: Method-1 (standard method) is the recommended method for bi-directional sequencing using the LIB-A kit; Method-2 is a new option designed in this study for unidirectional sequencing with the LIB-A kit; and Method-3 uses the LIB-L kit for unidirectional sequencing. In our comparison among these three methods using 10 different environmental samples, Method-2 and Method-3 produced 1.5-1.6 times more useable reads than the standard method (Method-1), after quality-based trimming, and did not compromise the outcome of microbial community analyses. Specifically, Method-3 is the most cost-effective unidirectional amplicon sequencing method as it provided the most reads and required the least effort in consumables management. CONCLUSIONS: Our findings clearly demonstrated that alternative pyrosequencing methods for 16S rRNA genes could drastically affect sequencing output (e.g. number of reads before and after trimming) but have little effect on the outcomes of microbial community analysis. This finding is important for both researchers and sequencing facilities utilizing 16S rRNA gene pyrosequencing for microbial ecological studies.http://europepmc.org/articles/PMC3179495?pdf=render |
| spellingShingle | Hideyuki Tamaki Chris L Wright Xiangzhen Li Qiaoyan Lin Chiachi Hwang Shiping Wang Jyothi Thimmapuram Yoichi Kamagata Wen-Tso Liu Analysis of 16S rRNA amplicon sequencing options on the Roche/454 next-generation titanium sequencing platform. PLoS ONE |
| title | Analysis of 16S rRNA amplicon sequencing options on the Roche/454 next-generation titanium sequencing platform. |
| title_full | Analysis of 16S rRNA amplicon sequencing options on the Roche/454 next-generation titanium sequencing platform. |
| title_fullStr | Analysis of 16S rRNA amplicon sequencing options on the Roche/454 next-generation titanium sequencing platform. |
| title_full_unstemmed | Analysis of 16S rRNA amplicon sequencing options on the Roche/454 next-generation titanium sequencing platform. |
| title_short | Analysis of 16S rRNA amplicon sequencing options on the Roche/454 next-generation titanium sequencing platform. |
| title_sort | analysis of 16s rrna amplicon sequencing options on the roche 454 next generation titanium sequencing platform |
| url | http://europepmc.org/articles/PMC3179495?pdf=render |
| work_keys_str_mv | AT hideyukitamaki analysisof16srrnaampliconsequencingoptionsontheroche454nextgenerationtitaniumsequencingplatform AT chrislwright analysisof16srrnaampliconsequencingoptionsontheroche454nextgenerationtitaniumsequencingplatform AT xiangzhenli analysisof16srrnaampliconsequencingoptionsontheroche454nextgenerationtitaniumsequencingplatform AT qiaoyanlin analysisof16srrnaampliconsequencingoptionsontheroche454nextgenerationtitaniumsequencingplatform AT chiachihwang analysisof16srrnaampliconsequencingoptionsontheroche454nextgenerationtitaniumsequencingplatform AT shipingwang analysisof16srrnaampliconsequencingoptionsontheroche454nextgenerationtitaniumsequencingplatform AT jyothithimmapuram analysisof16srrnaampliconsequencingoptionsontheroche454nextgenerationtitaniumsequencingplatform AT yoichikamagata analysisof16srrnaampliconsequencingoptionsontheroche454nextgenerationtitaniumsequencingplatform AT wentsoliu analysisof16srrnaampliconsequencingoptionsontheroche454nextgenerationtitaniumsequencingplatform |