Single nucleotide polymorphism discovery in cutthroat trout subspecie... - SRA

SRX207944: Single nucleotide polymorphism discovery in cutthroat trout subspecies using genome reduction, barcoding, and 454 pyro-sequencing
1 LS454 (454 GS FLX Titanium) run: 1.4M spots, 731.7M bases, 1.6Gb downloads

Design: 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

Submitted by: Brigham Young University

Study: O. c. utah O. c. clarki O. c. pleuriticus O. c. stomias O. c. henshawi O. c. humboldtensis O. c. virginalis O. c. lewisi O. c. bouvieri Oncorhynchus mykiss Genome Sequencing

PRJNA182193 • SRP017383 • All experiments • All runs

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Single nucleotide polymorphism discovery in cutthroat trout subspecies using genome reduction, barcoding, and 454 pyro-sequencing

Sample: Single nucleotide polymorphism discovery in cutthroat trout subspecies using genome reduction, barcoding, and 454 pyro-sequencing

SAMN01821855 • SRS377845 • All experiments • All runs

Organism: Oncorhynchus clarkii

Library:

Instrument: 454 GS FLX Titanium

Strategy: OTHER

Source: GENOMIC

Selection: Reduced Representation

Layout: SINGLE