title: Identifying the plant-associated microbiome across aquatic and terrestrial environments: the effects of amplification method on taxa discovery.
abstract: Plants in terrestrial and aquatic environments contain a diverse microbiome. Yet, the chloroplast and mitochondria organelles of the plant eukaryotic cell originate from free-living cyanobacteria and Rickettsiales. This represents a challenge for sequencing the plant microbiome with universal primers, as ~99% of 16S rRNA sequences may consist of chloroplast and mitochondrial sequences. Peptide nucleic acid clamps offer a potential solution by blocking amplification of host-associated sequences. We assessed the efficacy of chloroplast and mitochondria-blocking clamps against a range of microbial taxa from soil, freshwater and marine environments. While we found that the mitochondrial blocking clamps appear to be a robust method for assessing animal-associated microbiota, Proteobacterial 16S rRNA binds to the chloroplast-blocking clamp, resulting in a strong sequencing bias against this group. We attribute this bias to a conserved 14-bp sequence in the Proteobacteria that matches the 17-bp chloroplast-blocking clamp sequence. By scanning the Greengenes database, we provide a reference list of nearly 1500 taxa that contain this 14-bp sequence, including 48 families such as the Rhodobacteraceae, Phyllobacteriaceae, Rhizobiaceae, Kiloniellaceae and Caulobacteraceae. To determine where these taxa are found in nature, we mapped this taxa reference list against the Earth Microbiome Project database. These taxa are abundant in a variety of environments, particularly aquatic and semiaquatic freshwater and marine habitats. To facilitate informed decisions on effective use of organelle-blocking clamps, we provide a searchable database of microbial taxa in the Greengenes and Silva databases matching various n-mer oligonucleotides of each PNA sequence.
authors.affiliation: Department of Ecology and Evolution, The University of Chicago, 1101 E 57th Street, Chicago, IL, 60637, USA.
authors.forename: Sara L
authors.initials: SL
authors.lastname: Jackrel
authors.affiliation: Biosciences Division, Argonne National Laboratory, 9700 S. Cass Avenue, Lemont, IL, 60439, USA.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Owens
authors.affiliation: Biosciences Division, Argonne National Laboratory, 9700 S. Cass Avenue, Lemont, IL, 60439, USA.|The Microbiome Center, Department of Surgery, The University of Chicago, 5841 S Maryland Ave, Chicago, IL, 60637, USA.
authors.forename: Jack A
authors.initials: JA
authors.lastname: Gilbert
authors.affiliation: Department of Ecology and Evolution, The University of Chicago, 1101 E 57th Street, Chicago, IL, 60637, USA.
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Pfister
mesh_terms: D001419:Bacteria / Q000145:classification* / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D056186:Metagenomics / Q000379:methods*; D064307:Microbiota; D010944:Plants / Q000382:microbiology*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S
keywords: Earth microbiome project; PNA clamps; Proteobacteria; aquatic environments; chloroplast; plant microbiome