http://lod.bco-dmo.org/id/dataset/662114
eng; USA
utf8
dataset
Highest level of data collection, from a common set of sensors or instrumentation, usually within the same research project
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
2016-10-20
ISO 19115-2 Geographic Information - Metadata - Part 2: Extensions for Imagery and Gridded Data
ISO 19115-2:2009(E)
V4 amplicon sequencing of coral tissue collected from Micronesia, the Florida Keys, and Australia in 2013 (Coral Microbial Relationships project)
2016-10-20
publication
2016-10-20
revision
BCO-DMO Linked Data URI
2016-10-20
creation
http://lod.bco-dmo.org/id/dataset/662114
Amy Apprill
Woods Hole Oceanographic Institution
principalInvestigator
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
publisher
Cite this dataset as: Apprill, A. (2016) V4 amplicon sequencing of coral tissue collected from Micronesia, the Florida Keys, and Australia in 2013 (Coral Microbial Relationships project). Biological and Chemical Oceanography Data Management Office (BCO-DMO). (Version 1) Version Date 2016-10-20 [if applicable, indicate subset used]. http://lod.bco-dmo.org/id/dataset/662114 [access date]
Accession numbers and locations of species analyzed. Dataset Description: <p>&nbsp;</p>
<p>Partial SSU rRNA genes of bacteria and archaea from tissue samples produced using 515F/806RB primers. Samples were collected in the Federated States of Micronesia, Austrailia, and the Florida&nbsp;Keys.</p> Methods and Sampling: <p><strong>Coral collection and processing</strong></p>
<p>Coral fragments were collected by a scuba diver using a hammer and chisel during field sampling trips to Kapangamarangi Atoll, Micronesia (November 2012), Florida Keys, U.S.A. (May 2013) and Magnetic Bay, Australia (November 2013). Fragments were stored in a cooler containing ice until they were flash frozen in liquid nitrogen. Fragments were obtained from 3 representative colonies of the following species:&nbsp;<em>Porites&nbsp;</em><em>lobata</em><em>,&nbsp;Pocillopora&nbsp;</em><em>verrucosa</em><em>,&nbsp;Acropora&nbsp;</em><em>humilis</em><em>, Orbicella&nbsp;</em><em>faveolata</em><em>, Montastraea&nbsp;</em><em>cavernosa</em><em>, O.&nbsp;</em><em>annularis</em><em>,&nbsp;and&nbsp;Diploria&nbsp;</em><em>strigosa</em>.&nbsp;Fragments were shipped back to Woods Hole Oceanographic Institution and stored at -80 C until they were processed.&nbsp;</p>
<p>Using an airbrush, an aerosolized jet of sterile 1-X phosphate-buffered-saline (PBS) was directed at fragments. This method physically separated the coral mucus and tissue from the skeleton and suspended the cellular material in a slurry. The slurry was homogenized and centrifuged at 4 C for 20 minutes (5000 rpm) to form cellular tissue pellets. The PBS supernatant was removed and the tissue was evenly divided into smaller sections using sterile razors. To ensure that differing DNA yields were solely attributed to the lysis efficacy of the extraction treatments, the amount of biomass entering each extraction was standardized for all samples (38.69 +/- 9.23 mg). Subsampled biomass fractions were stored in separate tubes and frozen at -80 C until they were extracted.&nbsp;</p>
<p><strong>Phase I DNA extractions&nbsp;</strong></p>
<p>DNA was extracted from the subsampled biomass using the PowerSoil&nbsp;(cat # 12888), PowerPlant&nbsp;Pro (cat # 13400), PowerBiofilm&nbsp;(cat # 24000), and UltraClean&nbsp;Tissue &amp; Cells (cat # 12334) DNA Isolation kits following the manufacturer’s instructions (Mo Bio Laboratories, Inc.). In this study, the treatments are referred to by their abbreviations: PowerSoil&nbsp;(PS), PowerPlant® Pro (PP), PowerBiofilm® (PB), and UltraClean&nbsp;Tissue &amp; Cells (UC). Manipulations to the mechanical lysis conditions for the UC extraction were made in phase II (see below), resulting in three permutations of the UC kit: Vortex Garnet (VG),&nbsp;Powerlyzer&nbsp;Glass (PG), and Vortex Glass (VGl).&nbsp; The optional proteinase-K digestion step (15 ul; 20 mg/mL at 60 C for 30 minutes) was implemented for all UC permutations. Samples were homogenized for 15 minutes using a vortex&nbsp;adaptor&nbsp;unless otherwise specified. Genomic DNA concentrations were assessed using the dsDNA&nbsp;High Sensitivity&nbsp;Qubit 2.0&nbsp;flourometric&nbsp;assay (Life Technologies).&nbsp;</p>
<p>DNA template was screened for PCR efficiency using the barcoded primer pair 515F and 806RB[36]. Unaltered DNA template (0.18 – 47 ng l-1) was amplified in 25 l reactions containing 1.25 U of GoTaq Flexi DNA Polymerase (Promega), 5X Colorless GoTaq Flexi Buffer, 2.5 mM MgCl2, 200 M dNTP mix (Promega), and 200 nM of each barcoded primer in a Bio-Rad thermocycler (Bio-Rad Laboratories). The following PCR reaction conditions were used: 95 C for 2 minutes, followed by 40 cycles of 95 C for 20 sec, 55 C for 15 sec, and 72 C for 5 minutes, concluding with an extension step of 72 C for 10 minutes. PCR products were visually screened electrophoretically for quality using a 1% agarose/TBE gel under UV light. Positive PCR amplification was denoted by the presence of an amplicon band approximately 292 bp in size. Band size was assessed using the Hyperladder 50 bp DNA ladder (5 ng l-1) (Bioline).&nbsp;</p>
<p><strong>Phase II: Mechanical lysis modifications</strong></p>
<p>The phase I extraction treatment yielding extracts with the highest PCR efficiency for all coral colonies and species (UC) was selected to further examine if differences in bead type and homogenization method and duration resulted in intra-treatment extraction biases (Table 1).&nbsp; DNA was extracted from the same coral colonies used during phase I using the UC kit with minor modifications to the mechanical lysis conditions. The average biomass per extraction was 42.26 +/- 13.95 mg. For the first modification, the garnet beads provided with the kit were replaced with 0.1 mm glass beads (cat # 13118, Mo Bio Laboratories, Inc.) (VGl). For the second modification, a PowerLyzer&nbsp;24 bench-top bead-based homogenizer (Mo Bio Laboratories, Inc., cat # 13155) was used to homogenize the tissue instead of the vortex adaptor and garnet beads were replaced with 0.1 mm glass beads (PG). Samples were homogenized with the&nbsp;Powerlyzer&nbsp;for 45 seconds at 3500 rpm. DNA was not extracted from three colonies (two&nbsp;<em>P.&nbsp;</em><em>verrucosa</em>&nbsp;and one&nbsp;<em>A.&nbsp;</em><em>humilis</em>)&nbsp;using the VGl treatment because of limited biomass. DNA concentrations were quantified and PCR efficiency assessed using the methods outlined above.</p>
<p><strong>Library preparation and sequencing&nbsp;</strong></p>
<p>Based on&nbsp;the phase&nbsp;I and II PCR screens, amplicons from the PB, VG, PG, and VGl extractions were selected for sequencing using the MiSeq platform (Illumina). In addition, two positive controls (<em>E. coli</em>&nbsp;DNA and the Human Microbiome Project mock community DNA (BEI Resources, cat # HM-276D)) and a negative control (U.V. sterilized DNA-free water) were amplified, barcoded, and included in the library pool. As an extra assessment of barcode reproducibility, each&nbsp;<em>O.&nbsp;</em><em>annularis</em>&nbsp;extract was assigned two unique barcodes, amplified in separate reactions, and sequenced.&nbsp;</p>
<p>DNA template was amplified with the same V4 primer set [36], using similar PCR reaction conditions as those described above, but with 35 cycles. Amplicons were purified using selective gel purification (MinElute PCR Purification Kit, Qiagen) so that only PCR products matching the approximate size of the V4 SSU rRNA gene amplicon were included in the final library pool. Samples were prepared for sequencing using the methods outlined by&nbsp;Apprill&nbsp;and colleagues [1]. The amplicon pool was shipped to the University of Illinois W. M. Keck Center for Comparative and Functional Genomics and sequenced using 2 x 250 bp paired-end MiSeq [1,2].&nbsp;</p>
<p><strong>References</strong></p>
<p>1. Apprill A, McNally S, Parsons R, Weber L. Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton. Aquat Microb Ecol. 2015;75(2):129-137; doi:10.3354/ame01753.&nbsp;</p>
<p>2.&nbsp; Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD. Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Appl Environ Microbiol.&nbsp;2013;79(17):5112-20; doi:10.1128/AEM.01043-13.</p>
<p><strong>Sequence processing&nbsp;</strong></p>
<p>Mothur&nbsp;software [1] (v.1.33.3) was used to combine the de-multiplexed paired reads (8,344,281 contigs) and remove longer sequences (&gt;275 bp) and sequences containing ambiguous base pairs. A subset of sequences with read lengths exceeding 275bp&nbsp;were&nbsp;queried using the NCBI&nbsp;BLASTN&nbsp;2.3.0 program [2,3]&nbsp;to evaluate top matches for these sequences. The remaining sequences were classified using the SILVA SSU Ref database [4] (v. 119) and sequences matching to Eukaryota, mitochondria, and ‘unknown’ lineages were discarded (2,802 sequences). Chloroplast sequences were retained to assess if more chloroplast sequences were associated with a particular DNA extraction treatment. The&nbsp;UCHIME&nbsp;algorithm [5] was used to identify and remove chimeric sequences (13,724 sequences total). Sequences were not subsampled [6,7].</p>
<p>The sequences were grouped using the Minimum Entropy Decomposition (MED) algorithm [8]. These MED nodes are analogous to operational taxonomic units (OTUs) and resolve biologically meaningful and distinct groups that can be separated by &lt;1% sequence disparity [8]. Taxonomy was assigned to MED nodes using the classify.seqs&nbsp;command in&nbsp;mothur&nbsp;[1] and the SILVA database [4] (v. 119). Sequences belonging to ‘unclassified’ MED nodes were re-aligned using the SINA alignment service [9] (v. 1.2.11)&nbsp;and imported into ARB [10] (SSURefNR99_123). Using ARB&nbsp;[10],&nbsp;phylogenetic comparisons were made between the aligned sequences and sequences&nbsp;within&nbsp;the SILVA database [4] (v. 119)&nbsp;using&nbsp;neighbor joining&nbsp;algorithms in an attempt to resolve ‘unclassified’ taxonomy.</p>
Funding provided by NSF Division of Ocean Sciences (NSF OCE) Award Number: OCE-1233612 Award URL: https://www.nsf.gov/awardsearch/show-award?AWD_ID=1233612
completed
Amy Apprill
Woods Hole Oceanographic Institution
508-289-2649
Marine Chemistry and Geochemistry Department 266 Woods Hole Road, MS# 4
Woods Hole
MA
02543
USA
aapprill@whoi.edu
pointOfContact
asNeeded
Dataset Version: 1
Unknown
coral_colony
coral_species
location
depth
lat
lon
UCT_CP_glass_accession
UCT_CP_glass_accessionLink
UCT_CP_glass_replicate_accession
UCT_CP_glass_replicate_accessionLink
UCT_CV_garnet_accession
UCT_CV_garnet_accessionLink
UCT_CV_garnet_replicate_accession
UCT_CV_garnet_replicate_accessionLink
PB_V_accession
PB_V_accessionLink
PB_V_replicate_accession
PB_V_replicate_accessionLink
UCT_CV_glass_accession
UCT_CV_glass_accessionLink
UCT_CV_glass_replicate_accession
UCT_CV_glass_replicate_accessionLink
Airbrush with 80 psi air pressure
PowerLyzer 24 Bench-Top Bead-Based Homogenizer
Bio-Rad thermocycler
theme
None, User defined
sample identification
species scientific name (binomial)
site
depth
latitude
longitude
accession number
featureType
BCO-DMO Standard Parameters
Airbrush
Homogenizer
Thermal Cycler
instrument
BCO-DMO Standard Instruments
Apprill_2013
service
Deployment Activity
Bermuda, Red Sea, Federated States of Micronesia, Florida Keys, and Australia
place
Locations
otherRestrictions
otherRestrictions
Access Constraints: none. Use Constraints: Please follow guidelines at: http://www.bco-dmo.org/terms-use Distribution liability: Under no circumstances shall BCO-DMO be liable for any direct, incidental, special, consequential, indirect, or punitive damages that result from the use of, or the inability to use, the materials in this data submission. If you are dissatisfied with any materials in this data submission your sole and exclusive remedy is to discontinue use.
Fundamental Coral-Microbial Associations
https://osprey.bco-dmo.org/project/564442
Fundamental Coral-Microbial Associations
<p><em>Description from NSF award abstract:</em><br />
Reef-building corals are in decline worldwide due in part to climate change and other human activities, and it is becoming increasingly important to understand what aspects of coral biology are degraded by environmental stress which then leads to coral mortality. It is now widely known that corals harbor communities of bacteria and archaea that are believed to play important roles in maintaining the health of their hosts, but we lack any appreciable understanding about the identity of the microbial associates regularly residing within healthy, reef-building corals. This project asks the central question: do reef-building corals harbor fundamental or persistent microbial associates that are symbiotic within their tissues? In order to address this hypothesis, the investigator will assess the identity of the bacterial and archaeal microbes using a variety of molecular and microscopy approaches that includes the identification and localization of a widespread group of coral bacterial associates belonging to the genus Endozoicomonas. The results of this study will then be used to develop additional questions about the role of these microbial associates in nutrient cycling and how they contribute to the health and survival of corals.</p>
Coral Microbial Relationships
largerWorkCitation
project
eng; USA
oceans
Bermuda, Red Sea, Federated States of Micronesia, Florida Keys, and Australia
-81.39111
146.852
-15.0883
24.606054
2016-10-20
Florida Keys, Federated States of Micronesia, Red Sea, & Bermuda
0
BCO-DMO catalogue of parameters from V4 amplicon sequencing of coral tissue collected from Micronesia, the Florida Keys, and Australia in 2013 (Coral Microbial Relationships project)
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
http://lod.bco-dmo.org/id/dataset-parameter/662126.rdf
Name: coral_colony
Units: unitless
Description: <p>PI issued ID for the coral colony sampled</p>
http://lod.bco-dmo.org/id/dataset-parameter/662127.rdf
Name: coral_species
Units: unitless
Description: <p>Species of coral sampled</p>
http://lod.bco-dmo.org/id/dataset-parameter/662128.rdf
Name: location
Units: unitless
Description: <p>Geographic location where sampling occurred</p>
http://lod.bco-dmo.org/id/dataset-parameter/662129.rdf
Name: depth
Units: meters
Description: <p>Depth where sampling occurred</p>
http://lod.bco-dmo.org/id/dataset-parameter/662130.rdf
Name: lat
Units: decimal degrees
Description: <p>Latitude; N is positive</p>
http://lod.bco-dmo.org/id/dataset-parameter/662131.rdf
Name: lon
Units: decimal degrees
Description: <p>Longitude; W is positive</p>
http://lod.bco-dmo.org/id/dataset-parameter/662132.rdf
Name: UCT_CP_glass_accession
Units: unitless
Description: <p>Accession number for DNA extracted from subsampled biomass using Ultra Clean tissue and cells (UC) Isolation Kit; manipulation to the mechanical lysis condition</p>
http://lod.bco-dmo.org/id/dataset-parameter/662133.rdf
Name: UCT_CP_glass_accessionLink
Units: unitless
Description: <p>Accession link for DNA extracted from subsampled biomass using Ultra Clean tissue and cells (UC) Isolation Kit; manipulation to the mechanical lysis condition</p>
http://lod.bco-dmo.org/id/dataset-parameter/662134.rdf
Name: UCT_CP_glass_replicate_accession
Units: unitless
Description: <p>Replicate accession number for DNA extracted from subsampled biomass using Ultra Clean tissue and cells (UC) Isolation Kit; manipulation to the mechanical lysis condition</p>
http://lod.bco-dmo.org/id/dataset-parameter/662135.rdf
Name: UCT_CP_glass_replicate_accessionLink
Units: unitless
Description: <p>Replicate accession link for DNA extracted from subsampled biomass using Ultra Clean tissue and cells (UC) Isolation Kit; manipulation to the mechanical lysis condition.</p>
http://lod.bco-dmo.org/id/dataset-parameter/662136.rdf
Name: UCT_CV_garnet_accession
Units: unitless
Description: <p>Accession number for DNA extracted from subsampled biomass using Ultra Clean tissue and cells (UC) Isolation Kit; manipulation to the mechanical lysis condition Vortex garnet (VG) beads permutation.</p>
http://lod.bco-dmo.org/id/dataset-parameter/662137.rdf
Name: UCT_CV_garnet_accessionLink
Units: unitless
Description: <p>Accession link for DNA extracted from subsampled biomass using Ultra Clean tissue and cells (UC) Isolation Kit; manipulation to the mechanical lysis condition Vortex garnet (VG) beads permutation.</p>
http://lod.bco-dmo.org/id/dataset-parameter/662138.rdf
Name: UCT_CV_garnet_replicate_accession
Units: unitless
Description: <p>Replicate accession number for DNA extracted from subsampled biomass using Ultra Clean tissue and cells (UC) Isolation Kit; manipulation to the mechanical lysis condition Vortex garnet (VG) beads permutation.</p>
http://lod.bco-dmo.org/id/dataset-parameter/662139.rdf
Name: UCT_CV_garnet_replicate_accessionLink
Units: unitless
Description: <p>Replication accession link for DNA extracted from subsampled biomass using Ultra Clean tissue and cells (UC) Isolation Kit; manipulation to the mechanical lysis condition Vortex garnet (VG) beads permutation.</p>
http://lod.bco-dmo.org/id/dataset-parameter/662140.rdf
Name: PB_V_accession
Units: unitless
Description: <p>Accession number for DNA extracted from subsampled biomass using PowerBiofilm (PB) Isolation Kit</p>
http://lod.bco-dmo.org/id/dataset-parameter/662141.rdf
Name: PB_V_accessionLink
Units: unitless
Description: <p>Accession link for DNA extracted from subsampled biomass using PowerBiofilm (PB) Isolation Kit</p>
http://lod.bco-dmo.org/id/dataset-parameter/662142.rdf
Name: PB_V_replicate_accession
Units: unitless
Description: <p>Replicate accession number for DNA extracted from subsampled biomass using PowerBiofilm (PB) Isolation Kit</p>
http://lod.bco-dmo.org/id/dataset-parameter/662143.rdf
Name: PB_V_replicate_accessionLink
Units: unitless
Description: <p>Replicate accession link for DNA extracted from subsampled biomass using PowerBiofilm (PB) Isolation Kit</p>
http://lod.bco-dmo.org/id/dataset-parameter/662144.rdf
Name: UCT_CV_glass_accession
Units: unitless
Description: <p>Accession number for DNA extracted from subsampled biomass using Ultra Clean tissue and cells (UC) Isolation Kit; manipulation to the mechanical lysis condition Vortex glass (VGI) beads permutation.</p>
http://lod.bco-dmo.org/id/dataset-parameter/662145.rdf
Name: UCT_CV_glass_accessionLink
Units: unitless
Description: <p>Accession link for DNA extracted from subsampled biomass using Ultra Clean tissue and cells (UC) Isolation Kit; manipulation to the mechanical lysis condition Vortex glass (VGI) beads permutation.</p>
http://lod.bco-dmo.org/id/dataset-parameter/662146.rdf
Name: UCT_CV_glass_replicate_accession
Units: unitless
Description: <p>Replicate accession number for DNA extracted from subsampled biomass using Ultra Clean tissue and cells (UC) Isolation Kit; manipulation to the mechanical lysis condition Vortex glass (VGI) beads permutation.</p>
http://lod.bco-dmo.org/id/dataset-parameter/662147.rdf
Name: UCT_CV_glass_replicate_accessionLink
Units: unitless
Description: <p>Replicate accession link for DNA extracted from subsampled biomass using Ultra Clean tissue and cells (UC) Isolation Kit; manipulation to the mechanical lysis condition Vortex glass (VGI) beads permutation.</p>
GB/NERC/BODC > British Oceanographic Data Centre, Natural Environment Research Council, United Kingdom
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
11997
https://datadocs.bco-dmo.org/file/B11vAoWcAYJQLJ/coral_accessions.csv
coral_accessions.csv
Primary data file for dataset ID 662114
download
https://osprey.bco-dmo.org/dataset/662114/data/download
download
onLine
dataset
<p><strong>Coral collection and processing</strong></p>
<p>Coral fragments were collected by a scuba diver using a hammer and chisel during field sampling trips to Kapangamarangi Atoll, Micronesia (November 2012), Florida Keys, U.S.A. (May 2013) and Magnetic Bay, Australia (November 2013). Fragments were stored in a cooler containing ice until they were flash frozen in liquid nitrogen. Fragments were obtained from 3 representative colonies of the following species:&nbsp;<em>Porites&nbsp;</em><em>lobata</em><em>,&nbsp;Pocillopora&nbsp;</em><em>verrucosa</em><em>,&nbsp;Acropora&nbsp;</em><em>humilis</em><em>, Orbicella&nbsp;</em><em>faveolata</em><em>, Montastraea&nbsp;</em><em>cavernosa</em><em>, O.&nbsp;</em><em>annularis</em><em>,&nbsp;and&nbsp;Diploria&nbsp;</em><em>strigosa</em>.&nbsp;Fragments were shipped back to Woods Hole Oceanographic Institution and stored at -80 C until they were processed.&nbsp;</p>
<p>Using an airbrush, an aerosolized jet of sterile 1-X phosphate-buffered-saline (PBS) was directed at fragments. This method physically separated the coral mucus and tissue from the skeleton and suspended the cellular material in a slurry. The slurry was homogenized and centrifuged at 4 C for 20 minutes (5000 rpm) to form cellular tissue pellets. The PBS supernatant was removed and the tissue was evenly divided into smaller sections using sterile razors. To ensure that differing DNA yields were solely attributed to the lysis efficacy of the extraction treatments, the amount of biomass entering each extraction was standardized for all samples (38.69 +/- 9.23 mg). Subsampled biomass fractions were stored in separate tubes and frozen at -80 C until they were extracted.&nbsp;</p>
<p><strong>Phase I DNA extractions&nbsp;</strong></p>
<p>DNA was extracted from the subsampled biomass using the PowerSoil&nbsp;(cat # 12888), PowerPlant&nbsp;Pro (cat # 13400), PowerBiofilm&nbsp;(cat # 24000), and UltraClean&nbsp;Tissue &amp; Cells (cat # 12334) DNA Isolation kits following the manufacturer’s instructions (Mo Bio Laboratories, Inc.). In this study, the treatments are referred to by their abbreviations: PowerSoil&nbsp;(PS), PowerPlant® Pro (PP), PowerBiofilm® (PB), and UltraClean&nbsp;Tissue &amp; Cells (UC). Manipulations to the mechanical lysis conditions for the UC extraction were made in phase II (see below), resulting in three permutations of the UC kit: Vortex Garnet (VG),&nbsp;Powerlyzer&nbsp;Glass (PG), and Vortex Glass (VGl).&nbsp; The optional proteinase-K digestion step (15 ul; 20 mg/mL at 60 C for 30 minutes) was implemented for all UC permutations. Samples were homogenized for 15 minutes using a vortex&nbsp;adaptor&nbsp;unless otherwise specified. Genomic DNA concentrations were assessed using the dsDNA&nbsp;High Sensitivity&nbsp;Qubit 2.0&nbsp;flourometric&nbsp;assay (Life Technologies).&nbsp;</p>
<p>DNA template was screened for PCR efficiency using the barcoded primer pair 515F and 806RB[36]. Unaltered DNA template (0.18 – 47 ng l-1) was amplified in 25 l reactions containing 1.25 U of GoTaq Flexi DNA Polymerase (Promega), 5X Colorless GoTaq Flexi Buffer, 2.5 mM MgCl2, 200 M dNTP mix (Promega), and 200 nM of each barcoded primer in a Bio-Rad thermocycler (Bio-Rad Laboratories). The following PCR reaction conditions were used: 95 C for 2 minutes, followed by 40 cycles of 95 C for 20 sec, 55 C for 15 sec, and 72 C for 5 minutes, concluding with an extension step of 72 C for 10 minutes. PCR products were visually screened electrophoretically for quality using a 1% agarose/TBE gel under UV light. Positive PCR amplification was denoted by the presence of an amplicon band approximately 292 bp in size. Band size was assessed using the Hyperladder 50 bp DNA ladder (5 ng l-1) (Bioline).&nbsp;</p>
<p><strong>Phase II: Mechanical lysis modifications</strong></p>
<p>The phase I extraction treatment yielding extracts with the highest PCR efficiency for all coral colonies and species (UC) was selected to further examine if differences in bead type and homogenization method and duration resulted in intra-treatment extraction biases (Table 1).&nbsp; DNA was extracted from the same coral colonies used during phase I using the UC kit with minor modifications to the mechanical lysis conditions. The average biomass per extraction was 42.26 +/- 13.95 mg. For the first modification, the garnet beads provided with the kit were replaced with 0.1 mm glass beads (cat # 13118, Mo Bio Laboratories, Inc.) (VGl). For the second modification, a PowerLyzer&nbsp;24 bench-top bead-based homogenizer (Mo Bio Laboratories, Inc., cat # 13155) was used to homogenize the tissue instead of the vortex adaptor and garnet beads were replaced with 0.1 mm glass beads (PG). Samples were homogenized with the&nbsp;Powerlyzer&nbsp;for 45 seconds at 3500 rpm. DNA was not extracted from three colonies (two&nbsp;<em>P.&nbsp;</em><em>verrucosa</em>&nbsp;and one&nbsp;<em>A.&nbsp;</em><em>humilis</em>)&nbsp;using the VGl treatment because of limited biomass. DNA concentrations were quantified and PCR efficiency assessed using the methods outlined above.</p>
<p><strong>Library preparation and sequencing&nbsp;</strong></p>
<p>Based on&nbsp;the phase&nbsp;I and II PCR screens, amplicons from the PB, VG, PG, and VGl extractions were selected for sequencing using the MiSeq platform (Illumina). In addition, two positive controls (<em>E. coli</em>&nbsp;DNA and the Human Microbiome Project mock community DNA (BEI Resources, cat # HM-276D)) and a negative control (U.V. sterilized DNA-free water) were amplified, barcoded, and included in the library pool. As an extra assessment of barcode reproducibility, each&nbsp;<em>O.&nbsp;</em><em>annularis</em>&nbsp;extract was assigned two unique barcodes, amplified in separate reactions, and sequenced.&nbsp;</p>
<p>DNA template was amplified with the same V4 primer set [36], using similar PCR reaction conditions as those described above, but with 35 cycles. Amplicons were purified using selective gel purification (MinElute PCR Purification Kit, Qiagen) so that only PCR products matching the approximate size of the V4 SSU rRNA gene amplicon were included in the final library pool. Samples were prepared for sequencing using the methods outlined by&nbsp;Apprill&nbsp;and colleagues [1]. The amplicon pool was shipped to the University of Illinois W. M. Keck Center for Comparative and Functional Genomics and sequenced using 2 x 250 bp paired-end MiSeq [1,2].&nbsp;</p>
<p><strong>References</strong></p>
<p>1. Apprill A, McNally S, Parsons R, Weber L. Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton. Aquat Microb Ecol. 2015;75(2):129-137; doi:10.3354/ame01753.&nbsp;</p>
<p>2.&nbsp; Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD. Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Appl Environ Microbiol.&nbsp;2013;79(17):5112-20; doi:10.1128/AEM.01043-13.</p>
<p><strong>Sequence processing&nbsp;</strong></p>
<p>Mothur&nbsp;software [1] (v.1.33.3) was used to combine the de-multiplexed paired reads (8,344,281 contigs) and remove longer sequences (&gt;275 bp) and sequences containing ambiguous base pairs. A subset of sequences with read lengths exceeding 275bp&nbsp;were&nbsp;queried using the NCBI&nbsp;BLASTN&nbsp;2.3.0 program [2,3]&nbsp;to evaluate top matches for these sequences. The remaining sequences were classified using the SILVA SSU Ref database [4] (v. 119) and sequences matching to Eukaryota, mitochondria, and ‘unknown’ lineages were discarded (2,802 sequences). Chloroplast sequences were retained to assess if more chloroplast sequences were associated with a particular DNA extraction treatment. The&nbsp;UCHIME&nbsp;algorithm [5] was used to identify and remove chimeric sequences (13,724 sequences total). Sequences were not subsampled [6,7].</p>
<p>The sequences were grouped using the Minimum Entropy Decomposition (MED) algorithm [8]. These MED nodes are analogous to operational taxonomic units (OTUs) and resolve biologically meaningful and distinct groups that can be separated by &lt;1% sequence disparity [8]. Taxonomy was assigned to MED nodes using the classify.seqs&nbsp;command in&nbsp;mothur&nbsp;[1] and the SILVA database [4] (v. 119). Sequences belonging to ‘unclassified’ MED nodes were re-aligned using the SINA alignment service [9] (v. 1.2.11)&nbsp;and imported into ARB [10] (SSURefNR99_123). Using ARB&nbsp;[10],&nbsp;phylogenetic comparisons were made between the aligned sequences and sequences&nbsp;within&nbsp;the SILVA database [4] (v. 119)&nbsp;using&nbsp;neighbor joining&nbsp;algorithms in an attempt to resolve ‘unclassified’ taxonomy.</p>
Specified by the Principal Investigator(s)
<p><strong>Statistical analysis&nbsp;</strong></p>
<p>Data was tested for normality using the Shapiro-Wilk test. One-tailed t-tests were used for pair-wise comparisons after&nbsp;establishment&nbsp;of normality and equal variance. One-way ANOVAs were used for multiple group testing. If data failed normality, Friedman Repeated Measures Analysis of Variance (FRMANOVA) on ranks tests were conducted. When appropriate, Tukey’s, Holm-Sidak, or Dunn’s Method&nbsp;post-hoc&nbsp;tests were conducted to determine significantly different groups. P values ≤ 0.05 were accepted as being statistically significant. The above statistical tests were conducted using SigmaPlot software (v. 13).</p>
<p>Primer (v.7.0.9, Primer- E Ltd.) was used for prokaryotic community visualization and diversity analysis. Bray-Curtis distances were calculated from normalized, square root transformed sequence data and used to conduct non-metric multidimensional scaling (nMDS) and nested two- and one-way analysis of similarity (ANOSIM) tests. Presence/absence heat maps for&nbsp;O.&nbsp;faveolata,&nbsp;O.&nbsp;annularis, and&nbsp;A.&nbsp;humilis&nbsp;were created using the&nbsp;phyloseq&nbsp;[11] R package within the R-studio environment and a custom script [12] that was modified for this study.&nbsp; These heat maps were generated using distinct MED nodes that comprised 50% of all the reads obtained for each sample and thus represent the most dominant groups.&nbsp;Frequency&nbsp;of MED node detection was determined for each treatment and the percentage of detection agreement between pairwise treatments within each colony was assessed. One-tailed t-tests were used to reveal significantly different MED node detection between treatments.</p>
<p><strong>References</strong></p>
<p>1. Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD. Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Appl Environ Microbiol.&nbsp;2013;79(17):5112-20; doi:10.1128/AEM.01043-13.</p>
<p>2. Zhang Z, Schwartz S, Wagner L, Miller W. A greedy algorithm for aligning DNA sequences. J Comput Biol. 2000;7(1-2):203-14; doi:10.1089/10665270050081478.</p>
<p>3. Morgulis A, Coulouris G,&nbsp;Raytselia&nbsp;Y, Madden TL, Agarwala R, Schäffer AA. Database indexing for production MegaBLAST searches. Bioinformatics. 2008;24:1757-64; doi: 10.1093/bioinformatics/btn32.&nbsp;</p>
<p>4. Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glockner FO. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2013;41(D1):D590-D596; doi:10.1093/nar/gks1219.&nbsp;</p>
<p>5. Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R. UCHIME improves sensitivity and speed of chimera detection.&nbsp;Bioinformatics.&nbsp;2011;27(16):2194-200; doi: 10.1093/bioinformatics/btr381.&nbsp;</p>
<p>6. de&nbsp;Ca´rcer&nbsp; DA, Denman SE, McSweeney C, Morrison M. Evaluation of&nbsp;subsampling-based&nbsp;normalization strategies for tagged high-throughput sequencing data sets from gut microbiomes. Appl Environ Microbiol. 2011;77(24):8795–8798; doi: 10.1128/AEM.05491-11.</p>
<p>7. Holmes S, McMurdie PJ. Waste not, want not: why rarefying microbiome data is inadmissible. PLoS Comput Biol.&nbsp;2014;10(4):e1003531; doi:10.1371/journal.pcbi.1003531.&nbsp;</p>
<p>8. Eren AM, Maignien L, Sul WJ, Murphy LG, Grim SL, Morrison HG, Sogin ML. Oligotyping: differentiating between closely related microbial taxa using 16S rRNA gene data. Methods Ecol Evol. 2013;4:1111–1119; doi:10.1111/2041-210X.12114.</p>
<p>9. Pruesse E, Peplies J, Glöckner FO.&nbsp;SINA: accurate high-throughput multiple sequence alignment of ribosomal RNA genes. Bioinformatics. 2012;28:1823-1829; doi: 10.1093/bioinformatics/bts252. &nbsp;</p>
<p>10. Ludwig, W, Strunk O, Westram R, Richter L, Meier HY, Buchner A, Lai T, Steppi S, Jobb G, Förster W, Brettske I, Gerber S, Ginhart AW, Gross O, Grumann S, Hermann S, Jost R, König A, Liss T, Lüßmann R, May M, Nonhoff B, Reichel B, Strehlow R, Stamatakis A, Stuckmann N, Vilbig A, Lenke M, Ludwig T, Bode A,&nbsp;Schleifer KH. ARB: a software environment for sequence data. Nucleic Acids Res. 2004;32(4):1363-1371; doi:10.1093/nar/gkh293.&nbsp;</p>
<p>11. McMurdie PJ, Holmes S. Phyloseq: An R Package for reproducible interactive analysis and graphics of microbiome census data. PLoS ONE. 2013;8(4):e61217;&nbsp; doi:10.1371/journal.pone.0061217.&nbsp;</p>
<p>12. Neave MJ. MEDecomposition. GitHub repository.&nbsp;https://github.com/neavemj/globalCoralMicrobiomes. Accessed 15 November 2015.&nbsp;</p>
<p><strong>BCO-DMO Data Processing Notes:</strong></p>
<p>-removed spaces and replaced with underscores<br />
-reformatted column names to comply with BCO-DMO standards<br />
-converted all lat/lons&nbsp;to decimal degrees<br />
-added accession links<br />
-filled in blank cells with nd</p>
Specified by the Principal Investigator(s)
asNeeded
7.x-1.1
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
Airbrush with 80 psi air pressure
Airbrush with 80 psi air pressure
PI Supplied Instrument Name: Airbrush with 80 psi air pressure PI Supplied Instrument Description:Frozen coral tissue was removed from skeleton using airbrush. Instrument Name: Airbrush Instrument Short Name:Airbrush Instrument Description: Device for spraying liquid by means of compressed air.
PowerLyzer 24 Bench-Top Bead-Based Homogenizer
PowerLyzer 24 Bench-Top Bead-Based Homogenizer
PI Supplied Instrument Name: PowerLyzer 24 Bench-Top Bead-Based Homogenizer PI Supplied Instrument Description:Used to homogenize the coral tissue Instrument Name: Homogenizer Instrument Short Name:Homogenizer Instrument Description: A homogenizer is a piece of laboratory equipment used for the homogenization of various types of material, such as tissue, plant, food, soil, and many others.
Bio-Rad thermocycler
Bio-Rad thermocycler
PI Supplied Instrument Name: Bio-Rad thermocycler PI Supplied Instrument Description:Reactions carried out on a Bio-Rad thermocycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) Instrument Name: Thermal Cycler Instrument Short Name:Thermal Cycler Instrument Description: A thermal cycler or "thermocycler" is a general term for a type of laboratory apparatus, commonly used for performing polymerase chain reaction (PCR), that is capable of repeatedly altering and maintaining specific temperatures for defined periods of time. The device has a thermal block with holes where tubes with the PCR reaction mixtures can be inserted. The cycler then raises and lowers the temperature of the block in discrete, pre-programmed steps. They can also be used to facilitate other temperature-sensitive reactions, including restriction enzyme digestion or rapid diagnostics.
(adapted from http://serc.carleton.edu/microbelife/research_methods/genomics/pcr.html)
Deployment: Apprill_2013
Apprill_2013
BIOS
BIOS
laboratory
Apprill_2013
Amy Apprill
Woods Hole Oceanographic Institution
BIOS
BIOS
laboratory