Single nucleotide polymorphism discovery in cutthroat trout subspecies using  genome reduction, barcoding, and 454 pyro-sequencing
We followed the genomic reduction methodology described in detail by Maughan et al 2009.  In brief, genomic reduction was carried out using restriction enzymes EcoRI and BfaI to double digest genomic DNA at restriction sites that were conserved across all sub-species, and then attaching EcoRI and BfaI adapter sequences to the sticky ends using T4 DNA ligase.  Approximately 90% of the genome was then discarded through size exclusion via spin chromatography and biotin-streptavidin paramagnetic bead separation.  Prior to 454-pyrosequencing, unique MID-barcodes were added to the remaining restriction fragments for each individual sample via polymerase chain reaction (PCR).  DNA concentrations of the final PCR products were quantified using picogreen fluorescent dye, and then all PCR products were pooled in equimolar amounts to obtain a single sample totaling 5 µg of DNA.  Gel electrophoresis was performed on the pooled sample in a single lane of a 1.5% agarose gel, and DNA fragments ranging from ~450-600 base pairs (bp) in size were removed and then used for 454-pyrosequencing.  Sequencing was performed with Titanium reagents without DNA fragmentation on a 454 Life Sciences Genome Sequencer FLX located in the DNA Sequencing Center at Brigham Young University.  Samples were individually identified with the following multiplex identifiers:     MID Sequence Organism Number  MID1  ACGAGTGCGT 33801  MID10 TCTCTATGCG 92742  MID11 TGATACGTCT 134387  MID12 TACTGAGCTA 134410  MID13 CATAGTAGTG 130805  MID14 CGAGAGATAC 94736  MID15 ATACGACGTA 179073  MID16 TCACGTACTA 132038  MID17 CGTCTAGTAC 180022  MID18 TCTACGTAGC 131909  MID2  ACGCTCGACA 134304  MID20 ACGACTACAG 90414  MID21 CGTAGACTAG 96518  MID22 TACGAGTATG 57036  MID23 TACTCTCGTG 99839  MID24 TAGAGACGAG 99858  MID25 TCGTCGCTCG 90712  MID26 ACATACGCGT 239020  MID27 ACGCGAGTAT 239024  MID28 ACTACTATGT 187969  MID29 ACTGTACAGT 181526  MID3  AGACGCACTC 135225  MID30 AGACTATACT 136780  MID31 AGCGTCGTCT 133863  MID32 AGTACGCTAT 136834  MID33 ATAGAGTACT 187968  MID34 CACGCTACGT 142374  MID35 CAGTAGACGT 98251  MID36 CGACGTGACT 94217  MID37 TACACACACT 93748  MID4  AGCACTGTAG 179753  MID5  ATCAGACACG 135526  MID6  ATATCGCGAG 136684  MID7  CGTGTCTCTA 136733  MID8  CTCGCGTGTC 179631  MID9  TAGTATCAGC 91991