Vavouto Bay Metabarcoding

Collection Title:

Vavouto Bay Metabarcoding

Collection Description:

18S rDNA, 16S rDNA, and diatom 18S v4 rDNA amplicons from sediment DNA collected from Vavouto Bay New Caledonia.

Field of Research:

Environmental Impact Assessment

DOI:

https://doi.org/10.25919/5da80af25e87c

Start Date:

01 Jul 2017

End Date:

01 Jan 2019

Contact:Megan Gillmore
 Megan.Gillmore@csiro.au
Keywords:

metabarcoding Nickel eDNA sediment

Lineage:

The DNA was amplified using polymerase chain reaction (PCR) and three different sets of primers to target different components of the benthic community composition. The eukaryotic community was determined by PCR of the 18S rDNA gene, performed using the universal primers All18SF (5’-3’: TGGTGCATGGCCGTTCTTAGT) and All18SR (5’-3’: CATCTAAGGGCATCACAGACC) (Hardy et al., 2010). The prokaryotic community was determined by PCR of the 16S rDNA gene, performed using 515f (5′-3’: GTGYCAGCMGCCGCGGTAA) (Baker et al., 2003; Quince et al., 2011) and 806r (5′-3’: GGACTACNVGGGTWTCTAAT) (Apprill et al., 2015). The diatom community was determined by PCR of the SSU (18S V4) rDNA gene F- GCGGTAATTCCAGCTCCAATAG and R – CTCTGACAATGGAATACGAATA (Zimmerman et al., 2011) (Zimmermann et al., 2011). A summary of the thermal cycling profiles for each primer set is provided in supplementary Table S1. For all PCRs, amplification was performed in a total volume of 50 µL with 25 µL of AmpliTaq® Gold 360 Master Mix (Applied Biosystems, USA), 18 – 21 µL Water (Molecular Biology Reagent, Sigma Aldrich, USA), 1 µL of 10 µM forward and reverse primers, and 2 – 5 µL of genomic DNA. All PCRs were run with positive DNA controls: mussel (Mytilus) and polychaete (Capitellidae) DNA for eukaryotic 18S rDNA PCR, Halothermothrix for prokaryotic 16S rDNA PCR, and Phaeodactylum for the diatom specific PCR; and a negative control of 2 – 5 L of PCR grade Water (Molecular Biology Reagent, Sigma Aldrich, USA). Success of PCR amplification was confirmed using a microchip electrophoresis system (MultiNA, Shimadzu, Japan). PCR products were then pooled into a single tube (separate tube for each set of primers) and were then purified using the QIAquick PCR purification kit (Qiagen). The concentration and purity of the purified PCR products were determined as reported above for DNA. Sequencing was performed by the Ramaciotti Centre for Genomics (UNSW, Australia). The three pooled samples of 18S rDNA, 16S rDNA, and diatom 18S v4 rDNA amplicons were prepared with Illumina Tru-seq PCR-free library preparation kit and sequenced over one Illumina MiSeq run at 2x250 base-pair paired-end.

Credit:

Megan Gillmore

Licence:

Creative Commons Attribution-Noncommercial-No Derivatives 4.0 International Licence

Organisations:

CSIRO (Australia)

Attribution Statement:

Gillmore, Megan; Gissi, Francesca; Stauber, Jenny; Golding, Lisa; Stephenson, Sarah; Greenfield, Paul; Chariton, Anthony; Jolley, Dianne (2019): Vavouto Bay Metabarcoding. v1. CSIRO. Data Collection. https://doi.org/10.25919/5da80af25e87c

Rights Statement:

All Rights (including copyright) CSIRO 2019.

Access:

The metadata and files (if any) are available to the public.