Functional genes to assess nitrogen cycling and aromatic hydrocarbon degradation: primers and processing matter
Targeting sequencing to genes involved in key environmental processes, i.e. ecofunctional genes, provides an opportunity to sample nature’s gene guilds to greater depth and help link community structure to process-level outcomes. Vastly different approaches have been implemented for sequence process...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2013-09-01
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| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fmicb.2013.00279/full |
| _version_ | 1811283263148261376 |
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| author | Christopher Ryan Penton Timothy A Johnson John F Quensen Shoko eIwai James R Cole James M. Tiedje |
| author_facet | Christopher Ryan Penton Timothy A Johnson John F Quensen Shoko eIwai James R Cole James M. Tiedje |
| author_sort | Christopher Ryan Penton |
| collection | DOAJ |
| description | Targeting sequencing to genes involved in key environmental processes, i.e. ecofunctional genes, provides an opportunity to sample nature’s gene guilds to greater depth and help link community structure to process-level outcomes. Vastly different approaches have been implemented for sequence processing and, ultimately, for taxonomic placement of these gene reads. The overall quality of next generation sequence analysis of functional genes is dependent on multiple steps and assumptions of unknown diversity. To illustrate current issues surrounding amplicon read processing we provide examples for three ecofunctional gene groups. A combination of in-silico, environmental and cultured strain sequences was used to test new primers targeting the dioxin and dibenzofuran degrading genes dxnA1, dbfA1, and carAa. The majority of obtained environmental sequences were classified into novel sequence clusters, illustrating the discovery value of the approach. For the nitrite reductase step in denitrification, the well-known nirK primers exhibited deficiencies in reference database coverage, illustrating the need to refine primer-binding sites and/or to design multiple primers, while nirS primers exhibited bias against five phyla. Amino acid-based OTU clustering of these two N-cycle genes from soil samples yielded only 114 unique nirK and 45 unique nirS genus-level groupings, likely a reflection of constricted primer coverage. Finally, supervised and non-supervised OTU analysis methods were compared using the nifH gene of nitrogen fixation, with generally similar outcomes, but the clustering (non-supervised) method yielded higher diversity estimates and stronger site-based differences. High throughput amplicon sequencing can provide inexpensive and rapid access to nature’s related sequences by circumventing the culturing barrier, but each unique gene requires individual considerations in terms of primer design and sequence processing and classification. |
| first_indexed | 2024-04-13T02:08:19Z |
| format | Article |
| id | doaj.art-7abe51a958d54ee48469d6319b168184 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-13T02:08:19Z |
| publishDate | 2013-09-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-7abe51a958d54ee48469d6319b1681842022-12-22T03:07:23ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2013-09-01410.3389/fmicb.2013.0027959292Functional genes to assess nitrogen cycling and aromatic hydrocarbon degradation: primers and processing matterChristopher Ryan Penton0Timothy A Johnson1John F Quensen2Shoko eIwai3James R Cole4James M. Tiedje5Michigan State UniversityMichigan State UniversityMichigan State UniversityUniversity of CaliforniaMichigan State UniversityMichigan State UniversityTargeting sequencing to genes involved in key environmental processes, i.e. ecofunctional genes, provides an opportunity to sample nature’s gene guilds to greater depth and help link community structure to process-level outcomes. Vastly different approaches have been implemented for sequence processing and, ultimately, for taxonomic placement of these gene reads. The overall quality of next generation sequence analysis of functional genes is dependent on multiple steps and assumptions of unknown diversity. To illustrate current issues surrounding amplicon read processing we provide examples for three ecofunctional gene groups. A combination of in-silico, environmental and cultured strain sequences was used to test new primers targeting the dioxin and dibenzofuran degrading genes dxnA1, dbfA1, and carAa. The majority of obtained environmental sequences were classified into novel sequence clusters, illustrating the discovery value of the approach. For the nitrite reductase step in denitrification, the well-known nirK primers exhibited deficiencies in reference database coverage, illustrating the need to refine primer-binding sites and/or to design multiple primers, while nirS primers exhibited bias against five phyla. Amino acid-based OTU clustering of these two N-cycle genes from soil samples yielded only 114 unique nirK and 45 unique nirS genus-level groupings, likely a reflection of constricted primer coverage. Finally, supervised and non-supervised OTU analysis methods were compared using the nifH gene of nitrogen fixation, with generally similar outcomes, but the clustering (non-supervised) method yielded higher diversity estimates and stronger site-based differences. High throughput amplicon sequencing can provide inexpensive and rapid access to nature’s related sequences by circumventing the culturing barrier, but each unique gene requires individual considerations in terms of primer design and sequence processing and classification.http://journal.frontiersin.org/Journal/10.3389/fmicb.2013.00279/fullNitrogen FixationClustering analysisNitrogen CyclingnirK and nirSnifHaromatic hydrocarbon |
| spellingShingle | Christopher Ryan Penton Timothy A Johnson John F Quensen Shoko eIwai James R Cole James M. Tiedje Functional genes to assess nitrogen cycling and aromatic hydrocarbon degradation: primers and processing matter Frontiers in Microbiology Nitrogen Fixation Clustering analysis Nitrogen Cycling nirK and nirS nifH aromatic hydrocarbon |
| title | Functional genes to assess nitrogen cycling and aromatic hydrocarbon degradation: primers and processing matter |
| title_full | Functional genes to assess nitrogen cycling and aromatic hydrocarbon degradation: primers and processing matter |
| title_fullStr | Functional genes to assess nitrogen cycling and aromatic hydrocarbon degradation: primers and processing matter |
| title_full_unstemmed | Functional genes to assess nitrogen cycling and aromatic hydrocarbon degradation: primers and processing matter |
| title_short | Functional genes to assess nitrogen cycling and aromatic hydrocarbon degradation: primers and processing matter |
| title_sort | functional genes to assess nitrogen cycling and aromatic hydrocarbon degradation primers and processing matter |
| topic | Nitrogen Fixation Clustering analysis Nitrogen Cycling nirK and nirS nifH aromatic hydrocarbon |
| url | http://journal.frontiersin.org/Journal/10.3389/fmicb.2013.00279/full |
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