Computational protocol: Construction of a radiation hybrid panel and the first yellowtail (Seriola quinqueradiata) radiation hybrid map using a nanofluidic dynamic array

Similar protocols

Protocol publication

[…] Genotyping analysis was performed on 96 DNA samples including material from 93 hybrid RH cell lines. Yellowtail DNA and a mixture of yellowtail and mouse DNA were used as two independent positive controls. Mouse DNA alone was used as a negative control.For expressed sequences, total RNA purified from the brain, muscle, liver, heart, intestine, kidney, spleen, gonad, bladder, and gills of one immature yellowtail was sequenced using a Roche/454 FLX Pyrosequencer employing a next-generation sequencing technique with reference sequences [unpublished observations]. Furthermore, total RNA was prepared from 500 juvenile yellowtail and from the kidneys and gills of 100 young yellowtail individuals and sequenced using a Roche/454 FLX Pyrosequencer. Then, these sequences and the reference sequences were assembled and analyzed using a CLC Genomics Workbench (CLC bio, Aarhus, Denmark). Oligonucleotide primer pair DNA markers were derived from the yellowtail expressed sequences, and were designed using Primer 3 software [] (see Additional files and ).Genotyping was carried out using the BioMark™ HD system (Fluidigm, USA), which requires a pre-amplification reaction. Primer pairs for the pre-amplification PCR and genotyping reactions were designed based on the expression sequences of the ESTs. Pre-amplification PCR product size was designed to be in the range of 100–150 bp, and the second PCR product size in the BioMark™ HD system was designed to be in the range of 50–100 bp. Pre-amplification PCR was carried out using a Multiplex PCR Assay Kit (TaKaRa, Shiga, Japan) with 96 pooled primer pairs tested against each DNA sample (60 ng) in the RH panel, and against the positive and negative controls. Cycle conditions were as follows: a pre-dwell for 1 min at 94°C; 20 cycles of 30 s at 94°C, 90 s at 62°C, and 90 s at 72°C; and a post-dwell for 10 min at 72°C. The products of the pre-amplification PCR were diluted 1:5 with TE.Genotyping reactions were carried out on the Fluidigm platform using a BioMark™ 96.96 nanofluidic dynamic array (Fluidigm, USA) for gene expression analysis. A 5 μL sample mixture was prepared for each sample containing diluted pre-amplified DNA, TaqMan Gene Expression Master Mix (Applied Biosystems, CA, USA), EvaGreen (Biotium, CA, USA), and DNA Binding Sample Loading Reagent (Fluidigm, USA). An assay mix of 5 μL was prepared with each nested primer from the pre-amplification PCR, and Assay Loading Reagent (Fluidigm, USA). An IFC controller HX was used to prime the fluidic array with control line fluid, and then the samples and assay mix were combined in the inlets. After loading, the array was placed in the BioMark™ HD system for PCR under the following cycle conditions: a pre-dwell for 10 min at 95°C, 40 cycles of 15 s at 95°C, 10 s at 62°C, and 20 s at 72°C. The results were analyzed using the Real-Time PCR Analysis software, which is integrated into the BioMark™ HD system. [...] We used CarthaGene software to perform two-point linkage analyses and to determine marker order and inter-marker distances in centi Rays (cR). Linkage groups were determined using the group command at an LOD threshold of 4.0 and a distance threshold of 45, and referenced the genetic yellowtail linkage map [2, unpublished observations].EST alignments of linkage group 1 (SQ1) were performed with the TBLASTX algorithm searches against the cDNA database of zebrafish (Danio rerio), medaka (Oryzias latipes), and the green spotted pufferfish (Tetraodon nigroviridis) using Ensembl []. The sequences with the lowest E-value (< 1.0 × 10−5) were adopted in each EST alignment. […]

Pipeline specifications

Software tools MUSCLE, CLC Genomics Workbench, CarthaGene, TBLASTX
Applications Genome annotation, Nucleotide sequence alignment
Organisms Seriola quinqueradiata, Mus musculus, Oryzias latipes, Danio rerio