Genomestudio 2011

Blood samples were obtained from 188 individuals representing 8 different cattle breeds across a broad latitudinal range in China. These include Menggu cattle (MGC), Yanhuang cattle (YHC), Caidamu cattle (CDM), Pingwu cattle (PWC), Liangshan cattle (LSC), Zhaotong cattle (ZTC), Wenshan cattle (WSC), and Nandan cattle (NDC). Genomic DNA was extracted from blood samples using the TIANamp Blood DNA Kit (Tiangen Biotech Co. Ltd), and DNA with an A260/280 ratio ranging between 1.8 and 2.0 was subject to further analysis. In this study, we divided 8 breeds into 4 groups based on geographical locations (North group, Northwest group, Southwest group and South group) (Fig. 1). The genotyping platform adopted in this study was Illumina’s Infinium II Multi-Sample Assay. SNP chips were scanned using iScan and analyzed using Illumina’s GenomeStudio 2011. After filtering by the call rate of each given animal (threshold was > 95%), the final data including Log R Ratio (LRR) and B Allele Frequency (BAF) were exported from GenomeStudio software. To avoid the bias of population genetic estimation, genetic relationships between pairwise individuals were estimated using PI-HAT value implemented in PLINK v1.07^{82 (link)}, unrelated individuals with pairwise PI-HAT < 0.25 were kept for subsequent analyses.

Raw, probe level summary values as exported from Illumina GenomeStudio 2011 of Illumina HumanHT 12 V4 microarrays were imported into R using beadarray73 (link). Probes were background corrected followed by quantile normalization using the neqc command74 (link). The analysis was restricted to probes with a detection p-value of less than 0.01 in at least 10% of the samples and probes matching to GENCODE version 24 with at most 2 mismatches. A linear model was fitted using limma75 (link) to determine differential expression including parameters for gender, age, ethnicity and batch effects infection as well as the MGIT growth rate and array quality weights were incorporated76 (link) in order to account for between array quality differences. To account for between patient correlation, the duplicateCorrelation command from the limma package was used. All p-values were corrected for multiple hypothesis testing using the Benjamini-Hochberg procedure77 . Gene set enrichment analysis was carried out using the tmod package in R.

We sampled 316 Atlantic cod (Figure 1 and Table 1), consisting of 144 individuals from 5 locations from the Northwest Atlantic, 39 Frontal and 39 Coastal ecotype individuals from Iceland (classified by data storage tag (DST) profiles, see Pálsson and Thorsteinsson, 2003 ; Thorsteinsson et al., 2012 ), and 50 NEAC and 44 NCC individuals from the Northeast Atlantic.
DNA was extracted from muscle tissue using the E.Z.N.A Tissue DNA kit (Omega Bio-Tek, Norcross, GA, USA) and normalized to 100 ng μl⁻¹. All samples were individually genotyped using a 12K Illumina SNP chip for which 8165 SNPs were polymorphic in this data set, had a call rate of >95% and showed Mendelian inheritance in a separate set of individuals with a pedigree. Out of these SNPs, 602 were close to selected candidate genes, 1470 were nonsynonymous SNPs and the remaining 5857 SNPs were randomly distributed throughout the 23 different linkage groups (LGs). Genotype clustering was performed in Genome Studio 2011.1 (Illumina Inc., San Diego, CA, USA). The nomenclature of the LGs follows Hubert et al. (2010) (link) and the order of the SNPs are as in Berg et al. (2016) (link). All 8165 SNPs used were mapped to the published Atlantic cod genome (ATLCOD1C) (Star et al., 2011 (link)) in the same way as in Berg et al. (2016) (link) and details are available in dbSNP (www.ncbi.nlm.nih.gov/snp).

Genotyping was performed using the Illumina Golden Gate assay at the SNP&SEQ Technology Platform at Uppsala University, Sweden. A panel of 768 SNPs were genotyped following the service provider’s protocol. The SNPs assayed were chosen from a panel developed by Haseneyer et al. [36 (link)] (Additional file 2). Due to lack of mapping information at the time, SNPs were selected to represent different biological roles, as described in their annotation. Later obtained mapping information [38 ] for ~100 of the SNPs showed an even distribution among chromosomes. SNPs not fulfilling Illumina Golden Gate design recommendations were avoided. Results were analyzed using the software GenomeStudio 2011.1 (Illumina).