2007 Sydney Harbour Study
Marine sediment metagenome isolated from Sydney Harbor, Australia
Source DNA available from Dr Chris Hardy (Chris.Hardy@csiro.au) at CSIRO Entomology.  The aim of this study is to use a metagenomic approach to determine and contrast the diversity and occurrence of eukaryotic organisms in estuarine sediments from polluted and reference sites in Sydney Harbour, Australia. The methodology is broadly applicable as a tool for measuring the impact of environmental perturbation on eukaryotic biodiversity.    Environmental core samples were collected from 3 reference and 3 impacted locations (each location with 3 sites, each site with 4 replicates samples). Each replicate sample was further separated by sieving into 3 sieve-size fractions (macro >500um; meio 500-40um; and micro <40um). This created a total of 216 samples (108 reference samples, 108 impacted samples. DNA was extracted from each sample and conserved 18S primers were used to amplify a 200-300bp variable part in the 3' region of the eukaryotic 18S rRNA gene by PCR. 9 fusion primers with A and B adapters & 10 base barcodes were added by PCR to enable tracking of pooled 18S rDNA sequences back to 9 groups of 12 samples derived from the 3 reference locations and 3 sieve-size fractions. Use of a two gasket setup for 454 sequencing allowed us to use the same fusion primers for the corresponding 9 groups of 12 samples derived from the impacted locations/sieve-size fractions. Two samples were submitted to the Australian Genome Research Facility for 454 GS FLX sequencing, one Reference 18S amplicon library and one Impacted 18S amplicon library. 454 Primer A was used to sequence the 18S rDNA amplicons to ensure each barcode in the 9 forward fusion primers got read.   The SRA submission can be found using the Project Data link.