454 Methods
We created an EST library for L. goodei using 454 sequencing. This is part of a
larger endeavor to develop more genetic resources for the Lucania system which
includes research into visual ecology (eyes), color pattern polymorphism (fins),
speciation with respect to intrinsic genetic incompatibilities relative to its sister
species (ovaries and testes), adaptation to salinity (gills), and behavior (brains).
Hence, we created a pooled sample of RNA from fin, eye, gill, ovary, testes, and brain.
We used animals from five distinct populations across Florida (1- Upper Bridge
Wakulla River, Wakulla, Co., FL; 2- St. Mark’s National Wildlife Refuge Gambo Bayou,
Wakulla, Co., FL; 3- 26-Mile Bend, Everglades, Broward Co., FL; 4- Rum Island Park,
Santa Fe River, Columbia Co., FL; 5- Delk’s Bluff Bridge, Oklawaha River, Marion Co.,
FL). RNA was extracted using a standard Trizol protocol with phase-locked tubes,
and subsequently purified with sodium acetate/ethanol precipitaiton. Details on
cDNA synthesis/normalization, library preparation, and sequencing can be found in
Dassanayake et al. (2009). Preparation of barcoded GS FLX libraries from the
normalized cDNA samples, emulsion-based clonal amplification, and sequencing on
the 454 Genome Sequencer FLX system were performed in the W. M. Keck Center
for Comparative and Functional Genomics at the University of Illinois at UrbanaChampaign according to the manufacturer's instructions (454 Life Sciences,
Branford, CT).
We assembled the contigs using Newbler assembler (454 Life Sciences, Branford,
CT, USA). Assembly parameters were as follows: the minimum contig length was set
at 200 bp; the minimum overlap length was 60 bp; and the minimum overlap
identity was 95%. A total of large 29,838 contigs (> 300 bp) were generated by the
Newbler assembler.
Dassanayake M, Haas JS, Bohnert HJ, Cheeseman JM (2009) Shedding light on an
extremophile lifestyle through transcriptomics. New Phytologist 183, 764-775.