Computational protocol: Prion gene haplotypes of U.S. cattle

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Protocol publication

[…] Sequences from the 192 animals of the multi-breed beef and dairy panels were processed for polymorphism detection with Phred and Phrap [,], Polyphred 3.5 [], and Consed software []. A physical map linked to the PRNP consensus sequence and the location of polymorphisms was constructed in Vector NTI (v7.1). The map was annotated with all amplification and sequencing primers connected with the PRNP sequence. Replacement primers for those that hybridized to genomic loci containing polymorphisms were designed and used for additional amplification and sequencing of PRNP regions. PRNP nucleotide sequence with a phred score greater than 20 from at least two sequencing reads from the same animal was mapped to the corresponding nucleotide on reference sequence [GenBank:AJ298878]. Sequence compromised by SNP loci under associated amplification or sequencing primers was not analyzed for sequence coverage or the determination of genotypes. Regions reflecting poor sequence quality (<95% animal coverage) were identified, additional amplicons and sequencing primers were designed, and additional sequencing was performed. PRNP allele genotypes were mapped to reference sequence [GenBank:AJ298878] and stored in a relational database. A file of PRNP sequence annotated with all polymorphisms observed in this study and their frequencies in the beef diversity panel (MBCDP2.1) and dairy diversity panel (MDCP1.5) has been deposited in GenBank [GenBank:DQ457195]. [...] Unphased PRNP genotypes were assembled for each animal in datasets of the B. taurus, British, Continental, Holstein, and Composite of U.S. Brahman subgroups. Polymorphisms with more than two alleles, a minor allele frequency <0.05, or those not in Hardy-Weinberg equilibrium (Chi-square p < 0.01) were excluded from further analyses. Our cattle populations were not the result of random mating, violating an assumption of Hardy-Weinberg equilibrium. However, the Hardy-Weinberg test facilitated the identification of common haplotypes within the subgroups by excluding polymorphisms where the minor allele was amplified in a particular breed, yet had a low overall frequency.The extent of LD between the PRNP alleles of each dataset was calculated with pairwise r2 values (Haploview v3.2 []). Regions of LD were determined through visual inspection of LD graphs. Haplotypes were inferred in Haploview using the EM algorithm and a minimal set of polymorphisms was identified that collectively tagged all observed haplotypes predicted on four or more chromosomes in one or more of the five subgroups. The htSNPs identified across the five subgroup datasets were combined into a single set of 19 htSNPS. The 19 htSNPs were used to infer haplotypes within the five subgroup datasets. Median-Joining networks of PRNP haplotypes were constructed in Network (v4.111)[]. […]

Pipeline specifications

Software tools PolyPhred, Consed, Haploview
Applications Sanger sequencing, GWAS
Organisms Bos taurus
Diseases Nervous System Diseases, Encephalopathy, Bovine Spongiform