Diazotroph Community Characterization via a High-Throughput nifH Amplicon Sequencing and Analysis Pipeline
The dinitrogenase reductase gene ( ) is the most widely established molecular marker for the study of nitrogen-fixing prokaryotes in nature. A large number of PCR primer sets have been developed for amplification, and the effective deployment of these approaches should be guided by a rapid, easy-to-...
Saved in:
| Published in: | Applied and environmental microbiology Vol. 84; no. 4 |
|---|---|
| Main Authors: | , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
United States
15.02.2018
|
| Subjects: | |
| ISSN: | 1098-5336, 1098-5336 |
| Online Access: | Get more information |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Abstract | The dinitrogenase reductase gene (
) is the most widely established molecular marker for the study of nitrogen-fixing prokaryotes in nature. A large number of PCR primer sets have been developed for
amplification, and the effective deployment of these approaches should be guided by a rapid, easy-to-use analysis protocol. Bioinformatic analysis of marker gene sequences also requires considerable expertise. In this study, we advance the state of the art for
analysis by evaluating
primer set performance, developing an improved amplicon sequencing workflow, and implementing a user-friendly bioinformatics pipeline. The developed amplicon sequencing workflow is a three-stage PCR-based approach that uses established technologies for incorporating sample-specific barcode sequences and sequencing adapters. Based on our primer evaluation, we recommend the Ando primer set be used with a modified annealing temperature of 58°C, as this approach captured the largest diversity of
templates, including paralog cluster IV/V sequences. To improve
sequence analysis, we developed a computational pipeline which infers taxonomy and optionally filters out paralog sequences. In addition, we employed an empirical model to derive optimal operational taxonomic unit (OTU) cutoffs for the
gene at the species, genus, and family levels. A comprehensive workflow script named TaxADivA (TAXonomy Assignment and DIVersity Assessment) is provided to ease processing and analysis of
amplicons. Our approach is then validated through characterization of diazotroph communities across environmental gradients in beach sands impacted by the Deepwater Horizon oil spill in the Gulf of Mexico, in a peat moss-dominated wetland, and in various plant compartments of a sugarcane field.
Nitrogen availability often limits ecosystem productivity, and nitrogen fixation, exclusive to prokaryotes, comprises a major source of nitrogen input that sustains food webs. The
gene, which codes for the iron protein of the nitrogenase enzyme, is the most widely established molecular marker for the study of nitrogen-fixing microorganisms (diazotrophs) in nature. In this study, a flexible sequencing/analysis pipeline, named TaxADivA, was developed for
amplicons produced by Illumina paired-end sequencing, and it enables an inference of taxonomy, performs clustering, and produces output in formats that may be used by programs that facilitate data exploration and analysis. Diazotroph diversity and community composition are linked to ecosystem functioning, and our results advance the phylogenetic characterization of diazotroph communities by providing empirically derived
similarity cutoffs for species, genus, and family levels. The utility of our pipeline is validated for diazotroph communities in a variety of ecosystems, including contaminated beach sands, peatland ecosystems, living plant tissues, and rhizosphere soil. |
|---|---|
| AbstractList | The dinitrogenase reductase gene (nifH) is the most widely established molecular marker for the study of nitrogen-fixing prokaryotes in nature. A large number of PCR primer sets have been developed for nifH amplification, and the effective deployment of these approaches should be guided by a rapid, easy-to-use analysis protocol. Bioinformatic analysis of marker gene sequences also requires considerable expertise. In this study, we advance the state of the art for nifH analysis by evaluating nifH primer set performance, developing an improved amplicon sequencing workflow, and implementing a user-friendly bioinformatics pipeline. The developed amplicon sequencing workflow is a three-stage PCR-based approach that uses established technologies for incorporating sample-specific barcode sequences and sequencing adapters. Based on our primer evaluation, we recommend the Ando primer set be used with a modified annealing temperature of 58°C, as this approach captured the largest diversity of nifH templates, including paralog cluster IV/V sequences. To improve nifH sequence analysis, we developed a computational pipeline which infers taxonomy and optionally filters out paralog sequences. In addition, we employed an empirical model to derive optimal operational taxonomic unit (OTU) cutoffs for the nifH gene at the species, genus, and family levels. A comprehensive workflow script named TaxADivA (TAXonomy Assignment and DIVersity Assessment) is provided to ease processing and analysis of nifH amplicons. Our approach is then validated through characterization of diazotroph communities across environmental gradients in beach sands impacted by the Deepwater Horizon oil spill in the Gulf of Mexico, in a peat moss-dominated wetland, and in various plant compartments of a sugarcane field.IMPORTANCE Nitrogen availability often limits ecosystem productivity, and nitrogen fixation, exclusive to prokaryotes, comprises a major source of nitrogen input that sustains food webs. The nifH gene, which codes for the iron protein of the nitrogenase enzyme, is the most widely established molecular marker for the study of nitrogen-fixing microorganisms (diazotrophs) in nature. In this study, a flexible sequencing/analysis pipeline, named TaxADivA, was developed for nifH amplicons produced by Illumina paired-end sequencing, and it enables an inference of taxonomy, performs clustering, and produces output in formats that may be used by programs that facilitate data exploration and analysis. Diazotroph diversity and community composition are linked to ecosystem functioning, and our results advance the phylogenetic characterization of diazotroph communities by providing empirically derived nifH similarity cutoffs for species, genus, and family levels. The utility of our pipeline is validated for diazotroph communities in a variety of ecosystems, including contaminated beach sands, peatland ecosystems, living plant tissues, and rhizosphere soil.The dinitrogenase reductase gene (nifH) is the most widely established molecular marker for the study of nitrogen-fixing prokaryotes in nature. A large number of PCR primer sets have been developed for nifH amplification, and the effective deployment of these approaches should be guided by a rapid, easy-to-use analysis protocol. Bioinformatic analysis of marker gene sequences also requires considerable expertise. In this study, we advance the state of the art for nifH analysis by evaluating nifH primer set performance, developing an improved amplicon sequencing workflow, and implementing a user-friendly bioinformatics pipeline. The developed amplicon sequencing workflow is a three-stage PCR-based approach that uses established technologies for incorporating sample-specific barcode sequences and sequencing adapters. Based on our primer evaluation, we recommend the Ando primer set be used with a modified annealing temperature of 58°C, as this approach captured the largest diversity of nifH templates, including paralog cluster IV/V sequences. To improve nifH sequence analysis, we developed a computational pipeline which infers taxonomy and optionally filters out paralog sequences. In addition, we employed an empirical model to derive optimal operational taxonomic unit (OTU) cutoffs for the nifH gene at the species, genus, and family levels. A comprehensive workflow script named TaxADivA (TAXonomy Assignment and DIVersity Assessment) is provided to ease processing and analysis of nifH amplicons. Our approach is then validated through characterization of diazotroph communities across environmental gradients in beach sands impacted by the Deepwater Horizon oil spill in the Gulf of Mexico, in a peat moss-dominated wetland, and in various plant compartments of a sugarcane field.IMPORTANCE Nitrogen availability often limits ecosystem productivity, and nitrogen fixation, exclusive to prokaryotes, comprises a major source of nitrogen input that sustains food webs. The nifH gene, which codes for the iron protein of the nitrogenase enzyme, is the most widely established molecular marker for the study of nitrogen-fixing microorganisms (diazotrophs) in nature. In this study, a flexible sequencing/analysis pipeline, named TaxADivA, was developed for nifH amplicons produced by Illumina paired-end sequencing, and it enables an inference of taxonomy, performs clustering, and produces output in formats that may be used by programs that facilitate data exploration and analysis. Diazotroph diversity and community composition are linked to ecosystem functioning, and our results advance the phylogenetic characterization of diazotroph communities by providing empirically derived nifH similarity cutoffs for species, genus, and family levels. The utility of our pipeline is validated for diazotroph communities in a variety of ecosystems, including contaminated beach sands, peatland ecosystems, living plant tissues, and rhizosphere soil. The dinitrogenase reductase gene ( ) is the most widely established molecular marker for the study of nitrogen-fixing prokaryotes in nature. A large number of PCR primer sets have been developed for amplification, and the effective deployment of these approaches should be guided by a rapid, easy-to-use analysis protocol. Bioinformatic analysis of marker gene sequences also requires considerable expertise. In this study, we advance the state of the art for analysis by evaluating primer set performance, developing an improved amplicon sequencing workflow, and implementing a user-friendly bioinformatics pipeline. The developed amplicon sequencing workflow is a three-stage PCR-based approach that uses established technologies for incorporating sample-specific barcode sequences and sequencing adapters. Based on our primer evaluation, we recommend the Ando primer set be used with a modified annealing temperature of 58°C, as this approach captured the largest diversity of templates, including paralog cluster IV/V sequences. To improve sequence analysis, we developed a computational pipeline which infers taxonomy and optionally filters out paralog sequences. In addition, we employed an empirical model to derive optimal operational taxonomic unit (OTU) cutoffs for the gene at the species, genus, and family levels. A comprehensive workflow script named TaxADivA (TAXonomy Assignment and DIVersity Assessment) is provided to ease processing and analysis of amplicons. Our approach is then validated through characterization of diazotroph communities across environmental gradients in beach sands impacted by the Deepwater Horizon oil spill in the Gulf of Mexico, in a peat moss-dominated wetland, and in various plant compartments of a sugarcane field. Nitrogen availability often limits ecosystem productivity, and nitrogen fixation, exclusive to prokaryotes, comprises a major source of nitrogen input that sustains food webs. The gene, which codes for the iron protein of the nitrogenase enzyme, is the most widely established molecular marker for the study of nitrogen-fixing microorganisms (diazotrophs) in nature. In this study, a flexible sequencing/analysis pipeline, named TaxADivA, was developed for amplicons produced by Illumina paired-end sequencing, and it enables an inference of taxonomy, performs clustering, and produces output in formats that may be used by programs that facilitate data exploration and analysis. Diazotroph diversity and community composition are linked to ecosystem functioning, and our results advance the phylogenetic characterization of diazotroph communities by providing empirically derived similarity cutoffs for species, genus, and family levels. The utility of our pipeline is validated for diazotroph communities in a variety of ecosystems, including contaminated beach sands, peatland ecosystems, living plant tissues, and rhizosphere soil. |
| Author | Valderrama-Aguirre, Lina C Green, Stefan J Rishishwar, Lavanya Kostka, Joel E Gaby, John Christian Jordan, I King Valderrama-Aguirre, Augusto |
| Author_xml | – sequence: 1 givenname: John Christian surname: Gaby fullname: Gaby, John Christian organization: School of Biology, The Georgia Institute of Technology, Atlanta, Georgia, USA – sequence: 2 givenname: Lavanya surname: Rishishwar fullname: Rishishwar, Lavanya organization: PanAmerican Bioinformatics Institute, Cali, Valle del Cauca, Colombia – sequence: 3 givenname: Lina C surname: Valderrama-Aguirre fullname: Valderrama-Aguirre, Lina C organization: School of Natural Resources and Environmental Engineering, PhD Program in Sanitary and Environmental Engineering, Universidad del Valle, Cali, Valle del Cauca, Colombia – sequence: 4 givenname: Stefan J surname: Green fullname: Green, Stefan J organization: DNA Services Facility, Research Resources Center, University of Illinois at Chicago, Chicago, Illinois, USA – sequence: 5 givenname: Augusto surname: Valderrama-Aguirre fullname: Valderrama-Aguirre, Augusto organization: Regenerar, Center of Excellence for Regenerative and Personalized Medicine, Valle del Cauca, Colombia – sequence: 6 givenname: I King surname: Jordan fullname: Jordan, I King organization: PanAmerican Bioinformatics Institute, Cali, Valle del Cauca, Colombia – sequence: 7 givenname: Joel E surname: Kostka fullname: Kostka, Joel E email: joel.kostka@biology.gatech.edu organization: PanAmerican Bioinformatics Institute, Cali, Valle del Cauca, Colombia |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/29180374$$D View this record in MEDLINE/PubMed |
| BookMark | eNpNkM1LwzAchoNM3IfePEuOXjrz1bQ5ljmdMFFw9_Jbkq0ZbVKbVtj-egcqeHrfw8PDyztFIx-8ReiWkjmlLH8olq9zQlPKEppdoAklKk9SzuXoXx-jaYwHQoggMr9CY6ZoTngmJujw6OAU-i60FV6Ephm86494UUEHuredO0HvgsdfDjDgldtXyabqwrCv2qHH3u1WuGja2ukz82E_B-u183sM3uDCQ32MLuJ319raeXuNLndQR3vzmzO0eVpuFqtk_fb8sijWieaC9ck259QYutNCMAncGJkRq7URljEgIAQHSI1kQJUSJku3MoPMslwoSaiWbIbuf7RtF86DYl82Lmpb1-BtGGJJlVSKC0qyM3r3iw7bxpqy7VwD3bH8u4d9AxMMaVo |
| CitedBy_id | crossref_primary_10_1093_femsec_fiaa026 crossref_primary_10_1016_j_scitotenv_2021_145473 crossref_primary_10_1007_s00374_023_01756_y crossref_primary_10_3389_fmars_2021_754682 crossref_primary_10_1016_j_scitotenv_2024_169885 crossref_primary_10_5194_bg_17_851_2020 crossref_primary_10_1111_1462_2920_14480 crossref_primary_10_1042_BSR20201679 crossref_primary_10_1016_j_apsoil_2018_08_007 crossref_primary_10_1016_j_scitotenv_2019_134967 crossref_primary_10_1080_09593330_2020_1811390 crossref_primary_10_1016_j_apsoil_2021_104227 crossref_primary_10_1111_mec_16163 crossref_primary_10_3389_fmicb_2021_631244 crossref_primary_10_1016_j_soilbio_2023_109261 crossref_primary_10_1128_AEM_00443_19 crossref_primary_10_3389_fsufs_2021_617157 crossref_primary_10_1007_s00253_021_11629_9 crossref_primary_10_1111_jpy_13301 crossref_primary_10_3390_microorganisms10081564 crossref_primary_10_3390_microorganisms13020248 crossref_primary_10_1186_s12915_019_0710_0 crossref_primary_10_1016_j_biotechadv_2025_108580 crossref_primary_10_1002_lno_12324 crossref_primary_10_1111_gcb_14715 crossref_primary_10_3389_fmars_2022_875582 crossref_primary_10_3389_fmicb_2018_00703 crossref_primary_10_1038_s41598_019_55625_6 crossref_primary_10_1038_s41598_021_88380_8 crossref_primary_10_1038_s41396_021_01054_1 crossref_primary_10_3389_fmars_2022_868261 crossref_primary_10_5194_bg_17_5953_2020 crossref_primary_10_1093_femsre_fuae024 crossref_primary_10_3390_microorganisms12122634 crossref_primary_10_1016_j_scitotenv_2023_169740 crossref_primary_10_1128_spectrum_00516_24 crossref_primary_10_1007_s42832_020_0075_x crossref_primary_10_1038_s41598_021_85829_8 crossref_primary_10_1038_s41467_023_42481_2 crossref_primary_10_1016_j_foreco_2021_119464 crossref_primary_10_1093_femsec_fiaa208 crossref_primary_10_3389_fmicb_2020_01814 crossref_primary_10_3390_jof10040280 crossref_primary_10_1016_j_ibiod_2023_105723 crossref_primary_10_1016_j_watres_2020_116767 crossref_primary_10_1088_2515_7620_ac5a69 crossref_primary_10_3389_fmicb_2021_658668 crossref_primary_10_1111_jipb_13073 crossref_primary_10_1016_j_plaphy_2021_09_012 crossref_primary_10_1111_gcbb_12893 crossref_primary_10_1007_s00284_025_04250_w crossref_primary_10_1016_j_gca_2025_01_007 crossref_primary_10_1016_j_mimet_2023_106740 crossref_primary_10_1093_femsec_fiae095 crossref_primary_10_3390_microorganisms10020216 crossref_primary_10_3389_fmicb_2022_1063027 crossref_primary_10_1007_s11104_021_04883_3 crossref_primary_10_1007_s11368_024_03837_3 crossref_primary_10_1007_s00248_024_02460_6 crossref_primary_10_1016_j_pedsph_2022_07_007 crossref_primary_10_1128_AEM_00195_20 crossref_primary_10_1186_s12859_021_04555_0 crossref_primary_10_3389_fmicb_2021_675693 crossref_primary_10_1016_j_soilbio_2021_108286 crossref_primary_10_1016_j_geoderma_2020_114923 crossref_primary_10_3390_plants8100408 crossref_primary_10_1093_aob_mcz164 crossref_primary_10_3389_fmicb_2020_523142 |
| ContentType | Journal Article |
| Copyright | Copyright © 2018 American Society for Microbiology. |
| Copyright_xml | – notice: Copyright © 2018 American Society for Microbiology. |
| DBID | CGR CUY CVF ECM EIF NPM 7X8 |
| DOI | 10.1128/AEM.01512-17 |
| DatabaseName | Medline MEDLINE MEDLINE (Ovid) MEDLINE MEDLINE PubMed MEDLINE - Academic |
| DatabaseTitle | MEDLINE Medline Complete MEDLINE with Full Text PubMed MEDLINE (Ovid) MEDLINE - Academic |
| DatabaseTitleList | MEDLINE - Academic MEDLINE |
| Database_xml | – sequence: 1 dbid: NPM name: PubMed url: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed sourceTypes: Index Database – sequence: 2 dbid: 7X8 name: MEDLINE - Academic url: https://search.proquest.com/medline sourceTypes: Aggregation Database |
| DeliveryMethod | no_fulltext_linktorsrc |
| Discipline | Economics Engineering Biology |
| EISSN | 1098-5336 |
| ExternalDocumentID | 29180374 |
| Genre | Research Support, U.S. Gov't, Non-P.H.S Research Support, Non-U.S. Gov't Journal Article |
| GroupedDBID | --- -~X 0R~ 23M 2WC 39C 4.4 53G 5GY 5RE 5VS 6J9 85S AAGFI AAZTW ABOGM ABPPZ ACBTR ACGFO ACIWK ACNCT ACPRK ADBBV ADUKH AENEX AFRAH AGVNZ ALMA_UNASSIGNED_HOLDINGS AOIJS BAWUL BKOMP BTFSW CGR CS3 CUY CVF D0L DIK E.- E3Z EBS ECM EIF EJD F5P GX1 H13 HYE HZ~ K-O KQ8 L7B NPM O9- P2P PQQKQ RHI RNS RPM RSF RXW TAE TAF TN5 TR2 TWZ UHB W8F WH7 WOQ X6Y ~02 ~KM 7X8 |
| ID | FETCH-LOGICAL-c342t-b831dd1fc4426a3dd670eccd4e22a0a443aa5d62a1994d75b67a7e2849601c62 |
| IEDL.DBID | 7X8 |
| ISICitedReferencesCount | 86 |
| ISICitedReferencesURI | http://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=Summon&SrcAuth=ProQuest&DestLinkType=CitingArticles&DestApp=WOS_CPL&KeyUT=000423774600001&url=https%3A%2F%2Fcvtisr.summon.serialssolutions.com%2F%23%21%2Fsearch%3Fho%3Df%26include.ft.matches%3Dt%26l%3Dnull%26q%3D |
| ISSN | 1098-5336 |
| IngestDate | Thu Sep 04 18:16:56 EDT 2025 Mon Jul 21 05:55:19 EDT 2025 |
| IsDoiOpenAccess | false |
| IsOpenAccess | true |
| IsPeerReviewed | true |
| IsScholarly | true |
| Issue | 4 |
| Keywords | sequence analysis taxonomy nitrogen fixation next-generation sequencing metagenomics bioinformatics |
| Language | English |
| License | Copyright © 2018 American Society for Microbiology. |
| LinkModel | DirectLink |
| MergedId | FETCHMERGED-LOGICAL-c342t-b831dd1fc4426a3dd670eccd4e22a0a443aa5d62a1994d75b67a7e2849601c62 |
| Notes | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
| OpenAccessLink | https://aem.asm.org/content/aem/84/4/e01512-17.full.pdf |
| PMID | 29180374 |
| PQID | 1969934107 |
| PQPubID | 23479 |
| ParticipantIDs | proquest_miscellaneous_1969934107 pubmed_primary_29180374 |
| PublicationCentury | 2000 |
| PublicationDate | 2018-02-15 |
| PublicationDateYYYYMMDD | 2018-02-15 |
| PublicationDate_xml | – month: 02 year: 2018 text: 2018-02-15 day: 15 |
| PublicationDecade | 2010 |
| PublicationPlace | United States |
| PublicationPlace_xml | – name: United States |
| PublicationTitle | Applied and environmental microbiology |
| PublicationTitleAlternate | Appl Environ Microbiol |
| PublicationYear | 2018 |
| References | 31097421 - Appl Environ Microbiol. 2019 May 16;85(11) |
| References_xml | – reference: 31097421 - Appl Environ Microbiol. 2019 May 16;85(11): |
| SSID | ssj0004068 |
| Score | 2.532999 |
| Snippet | The dinitrogenase reductase gene (
) is the most widely established molecular marker for the study of nitrogen-fixing prokaryotes in nature. A large number of... The dinitrogenase reductase gene (nifH) is the most widely established molecular marker for the study of nitrogen-fixing prokaryotes in nature. A large number... |
| SourceID | proquest pubmed |
| SourceType | Aggregation Database Index Database |
| SubjectTerms | Bacteria - classification Bacteria - genetics Bacteria - metabolism Bacterial Physiological Phenomena Computational Biology DNA, Bacterial - genetics Ecosystem High-Throughput Nucleotide Sequencing - methods Metagenomics Microbiota - genetics Microbiota - physiology Nitrogen - metabolism Nitrogen Fixation Oxidoreductases - genetics Phylogeny Polymerase Chain Reaction Rhizosphere Sequence Analysis, DNA Soil Microbiology |
| Title | Diazotroph Community Characterization via a High-Throughput nifH Amplicon Sequencing and Analysis Pipeline |
| URI | https://www.ncbi.nlm.nih.gov/pubmed/29180374 https://www.proquest.com/docview/1969934107 |
| Volume | 84 |
| WOSCitedRecordID | wos000423774600001&url=https%3A%2F%2Fcvtisr.summon.serialssolutions.com%2F%23%21%2Fsearch%3Fho%3Df%26include.ft.matches%3Dt%26l%3Dnull%26q%3D |
| hasFullText | |
| inHoldings | 1 |
| isFullTextHit | |
| isPrint | |
| link | http://cvtisr.summon.serialssolutions.com/2.0.0/link/0/eLvHCXMwpV1JS8QwFH64oh5cxn0jgtc4bZqmzUkGFzzoMOAgcxvSJIP10FanCvrrfemiHjwIXnpKS8ny3pfke98HcBoFXImJZFTHQlJuw5hiGrGUceFpBLRBUIn6PNxG_X48GslBc-A2bWiVbUysArXJtTsj7zoZF4kh14vOi2fqXKPc7WpjoTEL8wFCGUfpikY_1MK9phROxhRhjWiJ7yzu9q7uzjyX7WhjVfYruKySzPXaf39vHVYbeEl69XzYgBmbdWCxNpx878BSW4c87cDKDynCTXi6TNVHXr7kxSNpikbKd3LxJedcV2uSt1QRRRw5hA5rh5_itSRZOrkhvYqbjm3ua3Y2fpaozJBW9oQM0sIVv9stGF5fDS9uaOPDQHXAWUmTOPCN8SeaYzpXgTEi8nDkDbeMKU9xHigVGsGU0xk2UZiISEUW8x7ujnwt2DbMZXlmd4EkUgm3J5YJtkxMKIX2jJGWG4xzvmZ7cNL27hinubu7UJnNX6fj7_7dg516iMZFrccxZtKPnYzO_h_ePoBlhDyx41374SHMT3CR2yNY0G9lOn05ruYPPvuDu0_5qs9X |
| linkProvider | ProQuest |
| openUrl | ctx_ver=Z39.88-2004&ctx_enc=info%3Aofi%2Fenc%3AUTF-8&rfr_id=info%3Asid%2Fsummon.serialssolutions.com&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.genre=article&rft.atitle=Diazotroph+Community+Characterization+via+a+High-Throughput+nifH+Amplicon+Sequencing+and+Analysis+Pipeline&rft.jtitle=Applied+and+environmental+microbiology&rft.au=Gaby%2C+John+Christian&rft.au=Rishishwar%2C+Lavanya&rft.au=Valderrama-Aguirre%2C+Lina+C&rft.au=Green%2C+Stefan+J&rft.date=2018-02-15&rft.eissn=1098-5336&rft.volume=84&rft.issue=4&rft_id=info:doi/10.1128%2FAEM.01512-17&rft_id=info%3Apmid%2F29180374&rft_id=info%3Apmid%2F29180374&rft.externalDocID=29180374 |
| thumbnail_l | http://covers-cdn.summon.serialssolutions.com/index.aspx?isbn=/lc.gif&issn=1098-5336&client=summon |
| thumbnail_m | http://covers-cdn.summon.serialssolutions.com/index.aspx?isbn=/mc.gif&issn=1098-5336&client=summon |
| thumbnail_s | http://covers-cdn.summon.serialssolutions.com/index.aspx?isbn=/sc.gif&issn=1098-5336&client=summon |