- Results: Read mapping accuracy did not differ notably between the ARS-UCD1.2 and UOA_Angus_1 assemblies. - We discovered and high-quality variants from ARS-UCD1.2 and UOA_Angus_1, respectively. - genotyped from UOA_Angus_1 than ARS-UCD1.2 alignments. - Using the composite likelihood ratio test, we detected 40 and 33 signatures of selection from ARS-UCD1.2 and UOA_Angus_1, respectively, but the overlap between both assemblies was low. - Conclusions: The ARS-UCD1.2 and UOA_Angus_1 assemblies are suitable for reference-guided genome analyses in Brown Swiss cattle. - Reference-quality assemblies are available for Here- ford (ARS-UCD1.2) [20], Angus (UOA_Angus_1) [17]. - The total length of the autosomes was bp for ARS-UCD1.2 and bp for UOA_Angus_1. - An average number of 295 ± 131 and 293 ± 130 million reads per sample aligned to autosomal sequences of ARS-UCD1.2 and UOA_Angus_1, respec- tively. - The slightly higher number of reads that mapped to ARS-UCD1.2 is likely due to its longer autosomal sequence. - for ARS-UCD1.2 and 82.29% for UOA_Angus_1) were flagged as duplicates.. - The mean percentage of high-quality reads was slightly higher for the ARS-UCD1.2 than UOA_Angus_1 autosomes but greater differences existed at some chromosomes. - The proportion of high-quality reads was higher for the ARS-UCD1.2 assembly than the UOA_Angus_1 assembly at 16 out of the 29 autosomes.. - The greatest difference was observed for chromosome 20, for which the proportion of high-quality reads was 2.03 percent points greater for the ARS-UCD1.2 assem- bly than the UOA_Angus_1 assembly (P = 4.5 x 10 -4. - Of and million reads that aligned to chromosome 20 of ARS-UCD1.2 and UOA_Angus_1, respectively and million were high-quality reads. - Among the 13 autosomes for which the percentage of high-quality reads was greater for the UOA_Angus_1 than ARS-UCD1.2 assembly, the greatest. - The mean coverage was between 13.76 (chromosome 19) and 14.45 (chromosome 27) for ARS-UCD1.2 and between 13.76 (chromosome 19) and 14.52 (chromosome 14) for UOA_Angus_1.. - For 32.40 and 33.80% of the high-quality variants, the genotype of at least one out of 161 BSW samples was missing using the ARS-UCD1.2 and UOA_Angus_1 align- ments, respectively. - Across all chromosomes, the number of missing genotypes was slightly higher (P = 0.087) for variants called from UOA_Angus_1 than ARS-UCD1.2 alignments. - At least one missing genotype was observed for 49.79 and 37.39% of the chromosome 12 variants for the UOA_Angus_1 and ARS-UCD1.2-called genotypes.. - Parameter Unit ARS-UCD1.2 UOA_Angus_1. - Summary statistics extracted from the BAM files after aligning the samples to either the ARS-UCD1.2 or UOA_Angus_1 assembly. - UOA_Angus . - 112 sequenced animals that had an average fold sequencing coverage of and when aligned to ARS-UCD1.2 and UOA_Angus_1, respectively, also had Illumina BovineHD array-called genotypes at 530,372 autosomal SNPs. - After imputation SNPs, 9,553 INDELs) and SNPs, 28,875 INDELs) vari- ants were fixed for the alternate allele in ARS-UCD1.2 and UOA_Angus_1, respectively (Supplementary File 3:. - Both the number and the percentage of vari- ants fixed for the alternate allele was higher (0.10 percent points the latter, P = 0.027) for the UOA_Angus_1 than the ARS-UCD1.2 assembly. - 22,488,261 and variants were polymorphic (i.e., minor allele count ≥ 1) among the 161 BSW ani- mals in ARS-UCD1.2 and UOA_Angus_1, respectively (Table 3). - The number of variants detected per sample ranged from 6.91 to 8.58 million in ARS- UCD1.2 and from 6.93 to 8.44 million in UOA_Angus_1. - More SNPs and INDELs were discovered for the ARS-UCD1.2 than UOA_Angus_1 assembly.. - overall variant and INDEL density was slightly higher for the ARS-UCD1.2 assembly, the SNP density was slightly higher for the UOA_Angus_1 assembly (Table 3).. - The number and density of high-quality variants seg- regating on the 29 autosomes was 2.04 (P = 0.51) and 0.45 (P = 0.39) percent points higher, respectively, for the ARS-UCD1.2 than the UOA_Angus_1 assembly (Fig. - Chromosomes 9 and 12 were the only autosomes for which more variants were detected using the UOA_Angus_1 than ARS-UCD1.2 assembly.. - The variant density of 26 out of the 29 autosomes (except for chromosomes 9, 12 and 26) was higher for the ARS-UCD1.2 assembly than the UOA_Angus_1 assembly.. - Number of variants detected on autosomes when the 161 BSW samples are aligned to the ARS-UCD1.2 (blue) and UOA_Angus_1 (orange) assembly. - Visual inspection revealed that the segment with an excess of polymor- phic sites was substantially larger in UOA_Angus_1 (7.6 Mb) than ARS-UCD1.2 (3.5 Mb) (Fig. - When the same metrics were calculated without chromosome 12, the average density of both SNPs and INDELs was higher for ARS-UCD1.2 than UOA_Angus_1 (Supplementary File 6: Table S4). - Segments with an excess of polymorphic sites were also detected on the ARS-UCD1.2 chromosomes Mb Mb Mb Mb), and 21 (20-21 Mb). - The strikingly higher number (+31%) of variants discovered at chromosome 28 for ARS- UCD1.2 than UOA_Angus_1 was due to an increased length of chromosome 28 in the ARS-UCD1.2 assembly (Fig. - Of and high-quality non- fixed variants and had more than two alleles in the ARS-UCD1.2 and UOA_Angus_1 alignments, respectively (Supplementary File 7: Table S5). - Most (69.75% for ARS-UCD1.2 and 69.09% for UOA_Angus_1) of the multi-allelic sites were INDELs. - However, the difference in percentage of multiallelic INDELs was 0.69 percent points higher (P = 2.55 x 10 -9 ) for UOA_Angus_1 than ARS-UCD1.2 autosomes.. - We observed and variants for ARS-UCD1.2 and UOA_Angus_1, respectively, for which the observed genotypes deviated significantly (P <. - The proportion of high-quality non- fixed variants for which the genotypes do not agree with Hardy-Weinberg proportions is 0.12 percent points higher for the UOA_Angus_1 than ARS-UCD1.2 assem- bly. - At chromosome 12, 3.29 percent points more vari- ants deviated from Hardy-Weinberg proportions for the UOA_Angus_1 than the ARS-UCD1.2 assembly (Supplementary File 8: Figure S3). - When variants located on chromosome 12 were excluded from this comparison, we observed and variants for the ARS-UCD1.2 and UOA_Angus_1 assembly, respectively, for which the observed genotypes deviated significantly (P <. - Using the VEP software, we predicted functional con- sequences based on the Ensembl genome annotation for and SNPs, and 2,931,222 and 2,843,257 INDELs, respectively, that were discov- ered from the ARS-UCD1.2 and UOA_Angus_1 align- ments. - Assembly ARS-UCD1.2 is displayed above the horizontal line (blue) and assembly UOA_Angus_1 is displayed below the horizontal line (orange). - ARS-UCD1.2 and UOA_Angus_1, respectively (Table 4, Supplementary File 9: Table S6). - Only 224,549 and and 1.35%) of the SNPs were in exons for ARS- UCD1.2 and UOA_Angus_1, respectively. - The majority of INDELs was in either intergenic (65.76% and 55.95%) or intronic regions (33.84% and 43.47%) for ARS-UCD1.2 and UOA_Angus_1, respectively (Table 4, Supplementary File 9: Table S6). - The percentage of SNPs and INDELs annotated to intergenic regions is 9.74 and 9.81 percent points higher, respectively, for the ARS-UCD1.2 than UOA_Angus_1 assembly. - In con- trast, the percentage of SNPs and INDELs annotated to intronic regions is 9.54 and 9.63 percent points higher, respectively, for the UOA_Angus_1 than the ARS- UCD1.2 assembly. - According to the Ensembl annota- tion of the autosomal sequences, intergenic, intronic and exonic regions span respectively and 3.80% in ARS-UCD1.2 and and 5.36% in UOA_Angus_1.. - ARS-UCD1.2 UOA_Angus_1. - The num- ber of variants with putatively high or moderate effects was higher for the UOA_Angus_1 than ARS-UCD1.2 assembly for 14 of 16 functional classes of annotations.. - alleles was not available, we considered ARS- UCD1.2) and UOA_Angus_1) sequence vari- ants that were either polymorphic or fixed for the alter- nate allele in the 161 BSW cattle. - The CLR test revealed 40 and 33 genomic regions (merged top 0.1% win- dows) encompassing ∼ 2.5 and ∼ 2.48 Mb, and 29 and 27 genes, respectively, from the ARS-UCD1.2 and the UOA_Angus_1 alignments (Fig. - CLR UOA_Angus_1. - UOA_Angus CLR UOA_Angus_1 = 657). - A putative selective sweep on chromosome 13 was identified using the ARS-UCD1.2 but not the UOA_Angus_1 assembly. - Selection signal distribution for both ARS-UCD1.2 (top panel) and UOA_Angus_1 assemblies (bottom panel). - This pat- tern indicates that the 161 sequenced BSW cattle carry a segment in the homozygous state that is more similar to the UOA_Angus_1 than ARS-UCD1.2 reference genome.. - The average imputation accu- racy (Beagle R 2 ) was median: 0.99) in the ARS-UCD1.2 and median: 0.99) in the UOA_Angus_1 assembly. - Following quality control and imputed vari- ants were respectively retained (with imputation accuracy of and for genetic investiga- tions in the ARS-UCD1.2 and UOA_Angus_1 dataset representing 56.75 and 56.54% of the and high-quality segregating variants. - The accuracy of imputation for the Phe279Tyr variant in the GHR gene was 0.92 and 0.88 for the ARS-UCD1.2 and UOA_Angus_1 assembly, respectively. - Manhattan plots showing association of sequence variants - imputed using ARS-UCD1.2 (blue and grey) and UOA_Angus_1 (orange and grey. - chromosome 20) is flipped between ARS-UCD1.2 and UOA_Angus_1. - The number (in thousands) of variants - imputed using ARS-UCD1.2 (blue) and UOA_Angus_1 (orange. - The SNP is located at and bp on the ARS-UCD1.2 and UOA_Angus_1 build (the ori- entation of chromosome 20 is flipped in UOA_Angus_1).. - Two adjacent SNPs (ARS-UCD1.2: g.611019G >. - UOA_Angus_1: g.81672806C >. - In 161 sequenced BSW cattle of our study, the alternate allele was detected in the heterozygous state in two and one animals using the ARS-UCD1.2 and UOA_Angus_1 datasets. - the mapping cohort, the lysine variant had a frequency of 0.0082 (Beagle R 2 : 0.98) and 0.0002 (Beagle R 2 : 0.82) in the ARS-UCD1.2 and UOA_Angus_1 imputed genotypes.. - Also, the DGAT1 gene was missing in the Ensembl annotation of the UOA_Angus_1 assembly.. - To investi- gate reference-guided sequence analyses from different assemblies, we aligned short sequencing reads of 161 BSW cattle to the Hereford-based ARS-UCD1.2 and Angus- based UOA_Angus_1 assemblies. - The sequence read mapping and variant genotyping accu- racy did not differ notably between the ARS-UCD1.2 and UOA_Angus_1 assemblies, indicating that both assem- blies are suitable for reference-guided genome analyses in BSW cattle. - How- ever, it is worth mentioning that the orientation of some chromosomes is flipped in UOA_Angus_1 (i.e., the begin- ning of the chromosome corresponds to the end in the corresponding ARS-UCD1.2 entry). - The number and density of INDELs that segregate in 161 BSW cattle was slightly lower when variants were called from the UOA_Angus_1 than ARS-UCD1.2 align- ment. - the UOA_Angus_1 than ARS-UCD1.2 alignment. - Both the ARS-UCD1.2 and UOA_Angus_1 assembly were constructed from PacBio continuous long reads. - While ARS-UCD1.2 was polished with short reads and manually curated, this step was not as extensively carried out for the UOA_Angus_1 assembly [17, 20]. - Our results may indi- cate that UOA_Angus_1 contains somewhat more arte- factual INDELs than ARS-UCD1.2. - The UOA_Angus_1 assembly lacks approxi- mately 9.5 million bases that likely correspond to the ARS- UCD1.2 chromosome 28 sequence from bp onwards. - According to the Ensembl (build 101) annota- tion of ARS-UCD1.2, this segment encompasses 67 genes that are consequently missing in the autosomal annotation of UOA_Angus_1.. - Differences in the functional annotations predicted for variants obtained from ARS-UCD1.2 and UOA_Angus_1 were evident from the output of the VEP tool. - The QTL encompassed DGAT1 using the ARS-UCD1.2 annotation. - However, DGAT1 was not annotated at the corresponding sequence of the UOA_Angus_1 assembly. - In fact, the ARS-UCD1.2 assembly is currently the widely accepted and universally applied bovine reference genome [44, 45]. - By visually inspecting the genes encompassed by the signa- tures of selection and manually lifting coordinates from ARS-UCD1.2 to UOA_Angus_1, we were able to confirm. - For instance, a strong selective sweep on chromosome 13 was only detected using the ARS-UCD1.2 assembly, while a puta- tive sweep on chromosome 22 was only detected using the UOA_Angus_1 assembly. - These variants were absent when the sequencing data were aligned to UOA_Angus_1, because their alternate alleles in ARS-UCD1.2 correspond to reference alleles in UOA_Angus_1. - Our results suggest that both the ARS-UCD1.2 and UOA_Angus_1 assembly are suitable for reference-guided genome analyses in BSW cattle. - The ARS-UCD1.2 assembly was created from a female cow, thus does not contain a Y chromosomal sequence. - For the sake of completeness, we expanded the ARS-UCD1.2 assembly with the Y chromosomal sequence from Btau 5.0 and the UOA_Angus_1 assembly with the X chromosomal sequence from ARS-UCD1.2.. - We considered ARS- UCD1.2) and UOA_Angus_1) biallelic SNP (segregating sites and SNP that were fixed for the non- reference allele) to calculate the CLR in 20 Kb windows with pre-computed empirical alternate allele frequency.. - Coordinates of the SNP were originally deter- mined according to the ARS-UCD1.2 build. - The imputed data with variants aligned to the ARS- UCD1.2 and UOA_Angus_1 assembly respectively, con- tained genotypes at and sequence variants.. - Number of variants detected per kilo base pair (Kb) along autosomal sequences of 161 BSW samples when aligned to the ARS-UCD1.2 (blue) and UOA_Angus_1 (orange) assembly.. - Assembly ARS-UCD1.2 is displayed in the top panel (blue) and assembly UOA_Angus_1 is displayed as a mirror image in the bottom panel (orange).. - Additional file 10: Table S7: Candidate selection signatures detected using ARS-UCD1.2 as reference. - Chromosome 22 region in ARS-UCD1.2 from Mb and corresponding region on UOA_Angus_1 between Mb with highlighted two selective sweep region
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