Exploiting public databases of genomic variation to quantify evolutionary constraint on the branch point sequence in 30 plant and animal species.

The branch point sequence is a degenerate intronic heptamer required for the assembly of the spliceosome during pre-mRNA splicing. Disruption of this motif may promote alternative splicing and eventually cause phenotype variation. Despite its functional relevance, the branch point sequence is not included in most genome annotations. Here, we predict branch point sequences in 30 plant and animal species and attempt to quantify their evolutionary constraints using public variant databases. We find an implausible variant distribution in the databases from 16 of 30 examined species. Comparative analysis of variants from whole-genome sequencing shows that variants submitted from exome sequencing or false positive variants are widespread in public databases and cause these irregularities. We then investigate evolutionary constraint with largely unbiased public variant databases in 14 species and find that the fourth and sixth position of the branch point sequence are more constrained than coding nucleotides. Our findings show that public variant databases should be scrutinized for possible biases before they qualify to analyze evolutionary constraint.

Characterization of a haplotype-reference panel for genotyping by low-pass sequencing in Swiss Large White pigs.

BACKGROUND: The key-ancestor approach has been frequently applied to prioritize individuals for whole-genome sequencing based on their marginal genetic contribution to current populations. Using this approach, we selected 70 key ancestors from two lines of the Swiss Large White breed that have been selected divergently for fertility and fattening traits and sequenced their genomes with short paired-end reads. RESULTS: Using pedigree records, we estimated the effective population size of the dam and sire line to 72 and 44, respectively. In order to assess sequence variation in both lines, we sequenced the genomes of 70 boars at an average coverage of 16.69-fold. The boars explained 87.95 and 95.35% of the genetic diversity of the breeding populations of the dam and sire line, respectively. Reference-guided variant discovery using the GATK revealed 26,862,369 polymorphic sites. Principal component, admixture and fixation index (FST) analyses indicated considerable genetic differentiation between the lines. Genomic inbreeding quantified using runs of homozygosity was higher in the sire than dam line (0.28 vs 0.26). Using two complementary approaches, we detected 51 signatures of selection. However, only six signatures of selection overlapped between both lines. We used the sequenced haplotypes of the 70 key ancestors as a reference panel to call 22,618,811 genotypes in 175 pigs that had been sequenced at very low coverage (1.11-fold) using the GLIMPSE software. The genotype concordance, non-reference sensitivity and non-reference discrepancy between thus inferred and Illumina PorcineSNP60 BeadChip-called genotypes was 97.60, 98.73 and 3.24%, respectively. The low-pass sequencing-derived genomic relationship coefficients were highly correlated (r > 0.99) with those obtained from microarray genotyping. CONCLUSIONS: We assessed genetic diversity within and between two lines of the Swiss Large White pig breed. Our analyses revealed considerable differentiation, even though the split into two populations occurred only few generations ago. The sequenced haplotypes of the key ancestor animals enabled us to implement genotyping by low-pass sequencing which offers an intriguing cost-effective approach to increase the variant density over current array-based genotyping by more than 350-fold.

Infertility due to defective sperm flagella caused by an intronic deletion in DNAH17 that perturbs splicing.

Artificial insemination in pig (Sus scrofa domesticus) breeding involves the evaluation of the semen quality of breeding boars. Ejaculates that fulfill predefined quality requirements are processed, diluted and used for inseminations. Within short time, eight Swiss Large White boars producing immotile sperm that had multiple morphological abnormalities of the sperm flagella were noticed at a semen collection center. The eight boars were inbred on a common ancestor suggesting that the novel sperm flagella defect is a recessive trait. Transmission electron microscopy cross-sections revealed that the immotile sperm had disorganized flagellar axonemes. Haplotype-based association testing involving microarray-derived genotypes at 41,094 SNPs of six affected and 100 fertile boars yielded strong association (P = 4.22 × 10-15) at chromosome 12. Autozygosity mapping enabled us to pinpoint the causal mutation on a 1.11 Mb haplotype located between 3,473,632 and 4,587,759 bp. The haplotype carries an intronic 13-bp deletion (Chr12:3,556,401-3,556,414 bp) that is compatible with recessive inheritance. The 13-bp deletion excises the polypyrimidine tract upstream exon 56 of DNAH17 (XM_021066525.1: c.8510-17_8510-5del) encoding dynein axonemal heavy chain 17. Transcriptome analysis of the testis of two affected boars revealed that the loss of the polypyrimidine tract causes exon skipping which results in the in-frame loss of 89 amino acids from DNAH17. Disruption of DNAH17 impairs the assembly of the flagellar axoneme and manifests in multiple morphological abnormalities of the sperm flagella. Direct gene testing may now be implemented to monitor the defective allele in the Swiss Large White population and prevent the frequent manifestation of a sterilizing sperm tail disorder in breeding boars.