Computational protocol: Maternal variant in the upstream of FOXP3 gene on the X chromosome is associated with recurrent infertility in Japanese Black cattle

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Protocol publication

[…] A total of 267 DNA samples were individually genotyped using the BovineSNP50K version 2 BeadChip (Illumina, Cat. #WG-450-2001), which includes probes for 54,609 SNPs. The UMD3.1 assembly of the bovine genome [] was used to map the positions of the SNPs. A total of 42,105 SNPs that passed our quality control criteria (call rate > 95%, minor allele frequency > 0.01, Hardy-Weinberg equilibrium P > 0.001 []), which were analyzed using PLINK v1.07 software [], were used for the association study. We performed a GWAS using the trait as a binary variable (i.e., repeat-breeding heifers that failed to conceive by following ET and control animals), as is commonly done in case-control studies. Association analyses were performed using GEMMA software [], which is based on a linear-mixed model approach using a genetic-relationship matrix estimated by SNP genotypes to model the correlation Sbetween the phenotypes of the sample subjects. [...] Haploview 4.2 software [] was used to analyze linkage disequilibria between the all SNPs that were located within or near identified QTLs. The diplotypes of the GWAS samples were estimated using Beagle 3.3.2 software [, ]. [...] Semen gDNA (200 ng/50 μL) samples from 517 Japanese Black sires (key ancestor sires of Japanese Black cattle) in low-EDTA TE buffer (Life Technologies, Cat. #12090–015) were individually sheared to 150–200 bp using Covarise (M & S Instruments Inc., Cat. #S220). Individual paired-end libraries were prepared and captured using a SureSelect XT Bovine All Exon (Agilent, Cat. #G9496B (#S-SN34)) [] and Bravo automated next-generation sequencing liquid handing system (Agilent) and sequenced on an Illumina HiSeq2500 sequencing system (2 × 101 bp). Initial sequence processing, base-calling, and de-multiplexed sequence reads were performed using Illumina CASAVA v.1.8.0. Sequence reads (FASTQ) for each sample were aligned to the UMD3.1 assembly of the bovine genome using Burrows-Wheeler Aligner []. Duplicates were marked using Picard software [], and indexed, merged, and sorted using SAMtools []. The UnifiedGenotyper and HaplotypeCaller tools of the GATK utility were used to identify and extract the SNPs and indels []. Functional variant annotation and effect prediction was performed using SnpEff [], PolyPhen-2 [], and Variant Effect Predictor (VEP) [] based on gene annotation of the UMD3.1 assembly of the bovine genome. [...] The FOXP3 exons and the upstream region were directly sequenced by the Sanger sequence method. Polymerase chain reaction (PCR) products using primer pairs are listed in Table S8 (Additional file ). PCR products were sequenced using the forward and reverse primers, respectively (Table S8) and the BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems), followed by electrophoresis using an ABI 3730 sequencer (Applied Biosystems) and SNP identification was performed using SeqScape software, V2.5 (Applied Biosystems). [...] Position-specific scoring matrices (PSSM), scoring scales, scores, and relative scores, for the neighboring g.92,377,635A > G sequence (± 20 bp) were analyzed with the JASPAR database []. Graphical representation of the frequency matrix was represented by SeqLogo []. […]

Pipeline specifications

Software tools PLINK, GEMMA, Haploview, BEAGLE
Application GWAS