Genomic DNA was isolated from ear tissue using a standard phenol/chloroform method and dissolved in Tris-EDTA buffer. DNA quality and concentration were determined using a Nanodrop-1000 spectrophotometer (Thermo Fisher, USA). A total of 336 Chinese Erhualian and 610 DLY pigs were genotyped for 61 565 SNPs using the Illumina PorcineSNP60K Beadchip according to the manufacturer’s protocol. Quality control procedures were carried out to remove SNPs with a call rate greater than 90%, a minor allele frequency (MAF) less than 0.01 and a significant deviation from Hardy-Weinberg equilibrium (P ≤ 10−5); moreover, animals with more than 10% missing genotypes or more than 5% Mendelian errors were removed from the dataset. A final set of 35 985 SNPs for 331 Chinese Erhualian and 56 216 SNPs for 610 DLY pigs were used for further statistical analysis. The large difference in number of SNPs that were retained for each population is explained by the fact SNPs on the Illumina PorcineSNP60K Beadchip were primarily ascertained based on a small panel of western pig genomes and that more SNPs had a MAF less than 0.01 for the Chinese Erhualian dataset than for western DLY dataset. SNP chromosomal positions were based on the current pig genome assembly (Sus Scrofa Build 10.2 assembly).
Porcine snp60k beadchip
Manufactured by Illumina
Sourced in United States
The Porcine SNP60K BeadChip is a laboratory equipment product designed for genetic analysis. It is a microarray-based genotyping platform that can simultaneously detect and analyze thousands of single nucleotide polymorphisms (SNPs) in the porcine genome.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Quantstudio 12k flex real time pcr system, by Thermo Fisher Scientific (2 mentions)
Genomestudio, by Illumina (1 mentions)
Infinium hd assay ultra, by Illumina (1 mentions)
Genomestudio software, by Illumina (1 mentions)
Snp genotyping platform, by Illumina (1 mentions)
Axiom porcine genotyping array, by Thermo Fisher Scientific (1 mentions)
Genechip porcine genome array, by Thermo Fisher Scientific (1 mentions)
Nanodrop 8000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Nanodrop 8000, by Thermo Fisher Scientific (1 mentions)
16 protocols using porcine snp60k beadchip
1
SNP genotyping in Chinese Erhualian and DLY pigs
2
Detecting CNVs in Porcine Genotypes
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All 120 samples were genotyped using a Porcine SNP60K BeadChip, and the raw data were extracted using GenomeStudio (Version 1.9.4, Illumina, Inc.). We used a strict quality control threshold (call rate > 98%, call frequency > 90%) to minimize the false-positive rate and eliminate low-quality samples. After quality control, no samples were excluded from CNV detection. The PennCNV software using a hidden Markov model (HMM), which allows detection of CNVs from Illumina or Affymetrix SNP chip data, was employed for CNV identification. PennCNV integrates various sets of information, including the total signal intensity of the log R Ratio (LRR), the distance between neighboring SNPs, the B allele frequency (BAF), the population frequency of the B allele (PFB) of SNPs, pedigree information, and the–gc model to adjust for “genomic waves” [11 (link), 43 , 65 (link), 66 (link)]. We could not obtain pedigree information for many of the samples because the samples were obtained from multiple locations across Congjiang County, China. Thus, pedigree information was not incorporated into the analysis. Moreover, the CNVs were aggregated using CNVRuler software [67 (link)].
3
Genotyping of Porcine Populations with Imputation and Quality Control
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A total of 12,200 animals (10,899 sows and 1,301 boars) across the four populations were genotyped with either the PorcineSNP60K Bead Chip (Illumina Inc., San Diego, CA, USA; 61,566 SNPs) or the GGP Porcine HD v1 80K (GeneSeek Inc., Neogen Co., Lincoln, NE, USA; 68,529 SNPs). The missing genotypes were imputed using the FImpute v2.2 software (Sargolzaei et al., 2014 (link)) to the 60K Bead chip. Quality control of genotype data consisted of removing SNPs with minor allele frequency lower than 0.05, SNP call rate below 0.90 and extreme deviation from Hardy-Weinberg equilibrium (with a p-value smaller than 10−6). The total number of individuals with genotypes involved in further analyses is reported in Table 1 . SPG populations had the largest number of individuals genotyped, with a total of 3,790 for SPG_LR and 5,271 for SPG_LR. TML populations had a lower number of individuals genotyped, with 1,587 for TML_LR and 1,552 for TML_YS. The final number of SNPs that passed the quality control was 48,911, 49,183, 44,824, and 44,875, for SPG_LR, SPG_LW, TML_LR, and TML_YS, respectively.
4
Genotyping and Quality Control of Pigs
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A total of 364 pigs, including their F0, F1 and F2 founder generations (72 animals), were genotyped with the Porcine SNP60K BeadChip [75] (link) following the Infinium HD Assay Ultra protocol (Illumina Inc.; San Diego, CA, USA) and the genotypes were visualized with the GenomeStudio software (Illumina Inc.; San Diego, CA, USA). The quality control of the 62,163 SNPs was performed by using Plink [76] (link) software removing markers with a minor allele frequency (MAF) <5% and animals with missing genotypes>5%. The SNP mapping and annotation was performed by using the pig assembly 10.2 [ftp://ftp.ncbi.hlm.nih.gov/genomes/Sus_scrofa/GEF/ ]. We also excluded markers which did not map in the Sscrofa10.2 version assembly. Pedstats program [77] (link) was used to check Mendelian inheritance errors.
5
Porcine SNP Genotyping and Quality Control
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DNA was extracted from each sample of ear tissue following the standard phenol/chloroform extraction method. All DNA samples were qualified and normalized to a final concentration of 50 ng/μL. DNA quality was assessed on the basis of the ratios of light absorption (A260/280 and A260/230) and electrophoresis. The quality and concentration of each genomic DNA sample met the requirements for the Illumina SNP genotyping platform. The 390 individuals were genotyped using the porcine SNP60K Beadchip of Illumina (San Diego, CA, USA) [12 (link)]. Quality control was performed using PLINK v 1.07 software [17 (link)]. SNP markers with genotype missing rates >0.05, call rate <95%, minor allele frequencies <0.01, and Hardy–Weinberg p≤10E-06 were excluded. Unmapped SNPS and SNPs located on sex chromosomes were removed in accordance with the Sus scrofa10.2 assembly of the reference genome [18 (link)]. Samples and SNPs that passed the filter were selected for subsequent GWA analysis.
6
Pig Traits and Genotypic Analysis
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Data from 144 pigs (25% Iberian × 75% Landrace), representing 26 full-sib families, from backcrossing five F1 males with 26 Landrace sows was utilized. Details about the management conditions and the phenotype information have been previously reported [65 (link)-67 (link)]. For this study and based on an previous principal components analysis [66 (link)] we selected 15 of the total 48 traits representing the most informative phenotypes within the dataset. Nine of the 15 traits were related to IMF fatty acid (FA) composition in LD muscle, seven correspond to indices of FA metabolism and the last one is the IMF percentage (Additional file 11 : Table S8). The Porcine SNP60K BeadChip (Illumina) [68 (link)] was used to genotype a total 197 pigs, including the 144 phenotyped animals and the founder population. Quality control excluded SNPs with minor allele frequency < 5% and with call rate < 95%. A subset of 48,119 SNPs were retained for subsequent analysis, in addition, previously detected polymorphisms in the MTTP, FABP4, FABP5, and ELOVL6 genes were also tested [67 (link),69 (link),70 (link)]. The genomic coordinates of the SNP correspond to the Sus scrofa genome sequence assembly (Sscrofa10.2, August 2011) [71 (link)] and were annotated using as reference the pig assembly 10.2 [ftp://ftp.ncbi.nlm.nih.gov/genomes/Sus_scrofa/GFF/ ].
7
Porcine SNP Genotyping and Analysis
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Animals from BC1_LD and BC1_PI were genotyped with Porcine SNP60K BeadChip (Illumina, San Diego, USA) and BC1_DU animals with Axiom Porcine Genotyping Array (Affymetrix, Inc.).
Common Single Nucleotide Polymorphisms (SNPs) in both arrays were mapped against the Sus scrofa 11.1 assembly and Plink software [19 (link)] was used afterwards to remove markers that showed a minor allele frequency (MAF) less than 5% and SNPs with more than 5% of missing genotypes. After filtering, a total of 38,424 SNPs were retained for association studies. In addition, the IGF2:g.3072G>A polymorphism was genotyped using a pyrosequencing protocol previously described [10 (link)] and a SNP located in the predicted 3’ UTR region of the gene (ENSSSCT00000039341.1:c.1469990C>T) was genotyped using Taqman OpenArrayTM genotyping plates custom-designed in a QuantStudioTM 12K flex Real-Time PCR System (ThermoFisher Scientific).
Common Single Nucleotide Polymorphisms (SNPs) in both arrays were mapped against the Sus scrofa 11.1 assembly and Plink software [19 (link)] was used afterwards to remove markers that showed a minor allele frequency (MAF) less than 5% and SNPs with more than 5% of missing genotypes. After filtering, a total of 38,424 SNPs were retained for association studies. In addition, the IGF2:g.3072G>A polymorphism was genotyped using a pyrosequencing protocol previously described [10 (link)] and a SNP located in the predicted 3’ UTR region of the gene (ENSSSCT00000039341.1:c.1469990C>T) was genotyped using Taqman OpenArrayTM genotyping plates custom-designed in a QuantStudioTM 12K flex Real-Time PCR System (ThermoFisher Scientific).
8
Genotyping and Quality Control of Porcine Samples
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Animals from BC1_LD and BC1_PI were genotyped using the Porcine SNP60K BeadChip (Illumina, San Diego, USA) and BC1_DU animals were genotyped using the GeneChip Porcine Genome Array (Affymetrix). Only SNPs that mapped against the Sscrofa11.1 assembly and were common to both arrays were selected35 (link). Markers that showed a minor allele frequency (MAF) lower than 5% and SNPs with more than 5% of missing genotypes were removed with Plink software66 (link). Moreover, based on the information in the prior BC1_LD study17 (link), two additional SNPs were genotyped: ACSM5 (rs331702081) and IGF2 (IGF2:g.3072 G > A), in the BC1_DU and BC1_PI populations, following the previously described protocols17 (link),22 (link). Finally, a total of 38.426 SNPs distributed along all chromosomes, including rs331702081 and IGF2:g.3072 G > A polymorphisms, were used for association studies.
9
Porcine Genotyping using SNP Chip
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All experimental animals in the LK cross were genotyped for 62,163 single nucleotide polymorphism (SNP) markers using the Porcine SNP 60K BeadChip (Illumina Inc., San Diego, CA, USA). Genomic DNA was extracted from the blood using a standard sucrose–proteinase K method. Quality control and filtration of genotyped SNP markers were performed using the PLINK program (ver. 1.90) [21 (link)]. The genotypes were filtered according to minor allele frequency (<5%), genotype call rate (<90%), and a p-value of the χ2-test for Hardy–Weinberg equilibrium errors (≤0.000001). A total of 39,485 SNP markers on 18 autosomes remained after filtration and quality control.
10
Indel Validation and Association Analysis in Porcine Backcrosses
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For indel validation and association analysis, ten indels were genotyped in three experimental backcrosses: BC1_DU (n = 143), BC1_LD (n = 160) and BC1_PI (n = 138), using Taqman OpenArray genotyping plates custom designed in a QuantStudio 12K flex Real-Time PCR System (ThermoFisher Scientific, Waltham, MA, USA).
The same animals of BC1_LD and BC1_PI were genotyped with the Porcine SNP60K BeadChip (Illumina, San Diego, CA, USA), while BC1_DU samples genotypes were obtained with the Axiom Porcine Genotyping Array (Axiom_PigHDv1; Affymetrix, Santa Clara, CA, USA). Only those variants shared by both genotyping platforms were kept. A total of 38,424 SNPs remained after removing SNPs with a minor allele frequency (MAF) < 5% and SNPs with missing genotype > 5% data using PLINK (1.90b5 version) [43 (link)].
The same animals of BC1_LD and BC1_PI were genotyped with the Porcine SNP60K BeadChip (Illumina, San Diego, CA, USA), while BC1_DU samples genotypes were obtained with the Axiom Porcine Genotyping Array (Axiom_PigHDv1; Affymetrix, Santa Clara, CA, USA). Only those variants shared by both genotyping platforms were kept. A total of 38,424 SNPs remained after removing SNPs with a minor allele frequency (MAF) < 5% and SNPs with missing genotype > 5% data using PLINK (1.90b5 version) [43 (link)].
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