Budget Total US: $4,550
9.2 Please
detail how you would spend the money you are requesting from the Species
Fund (200 word limit)
Money will be used to support field and laboratory work to purchase reagents, supplies and sequencing. Field work is relatively simple and inexpensive, but it requires that bottles are bleached beforehand, handled with gloves and stored on ice during transportation. Field work lasts for a single day, but it is repeated during Summer and Winter. In the laboratory, samples are filtered through cellulose filters and stored in foil in the -80°C freezer until they can be further processed. DNA extraction is done using a modification of the Qiagen Blood and Tissue protocol, and metabarcode libraries are generated for bacteria and archaea (16S), eukaryotes (18S), fungi (ITS), plants (ITS2), and metazoans (CO1) in triplicates using Qiagen Multiplex Mastermix and primers. Sequence libraries using the Nextera Index kit will then be set to run in a Illumina HiSeq 4000 with the goal of generating 300,000 reads per metabarcode library per sample. After this, reads will follow a pipeline that is currently under development by the CALeDNA program.
9.3 Please
specify if there is anything else you wish to highlight regarding this budget
(e.g. This funding would be combined with funding from other organizations) (200 word limit)
This research will receive partial aid from the CALeDNA program. They will provide the laboratory resources necessary to sample in the field and process the sediment and soil samples (up to 40 samples total). They have also been providing laboratory and bioinformatic training that will be applied for analysis of the water samples as well.