Anette Høj

SNP mining porcine ESTs with MAVIANT, a novel tool for SNP evaluation and annotation

MOTIVATION Single nucleotide polymorphisms (SNPs) analysis is an important means to study genetic variation. A fast and cost-efficient approach to identify large numbers of novel candidates is the SNP mining of large scale sequencing projects. The increasing availability of sequence trace data in public repositories makes it feasible to evaluate SNP predictions on the DNA chromatogram level. MAVIANT, a platform-independent Multipurpose Alignment VIewing and Annotation Tool, provides DNA chromatogram and alignment views and facilitates evaluation of predictions. In addition, it supports direct manual annotation, which is immediately accessible and can be easily shared with external collaborators. RESULTS Large-scale SNP mining of polymorphisms bases on porcine EST sequences yielded more than 7900 candidate SNPs in coding regions (cSNPs), which were annotated relative to the human genome. Non-synonymous SNPs were analyzed for their potential effect on the protein structure/function using the PolyPhen and SIFT prediction programs. Predicted SNPs and annotations are stored in a web-based database. Using MAVIANT SNPs can visually be verified based on the DNA sequencing traces. A subset of candidate SNPs was selected for experimental validation by resequencing and genotyping. This study provides a web-based DNA chromatogram and contig browser that facilitates the evaluation and selection of candidate SNPs, which can be applied as genetic markers for genome wide genetic studies. AVAILABILITY The stand-alone version of MAVIANT program for local use is freely available under GPL license terms at http://snp.agrsci.dk/maviant. SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

A robust linkage map of the porcine autosomes based on gene-associated SNPs

BackgroundGenetic linkage maps are necessary for mapping of mendelian traits and quantitative trait loci (QTLs). To identify the actual genes, which control these traits, a map based on gene-associated single nucleotide polymorphism (SNP) markers is highly valuable. In this study, the SNPs were genotyped in a large family material comprising more than 5,000 piglets derived from 12 Duroc boars crossed with 236 Danish Landrace/Danish Large White sows. The SNPs were identified in sequence alignments of 4,600 different amplicons obtained from the 12 boars and containing coding regions of genes derived from expressed sequence tags (ESTs) and genomic shotgun sequences.ResultsLinkage maps of all 18 porcine autosomes were constructed based on 456 gene-associated and six porcine EST-based SNPs. The total length of the averaged-sex whole porcine autosome was estimated to 1,711.8 cM resulting in an average SNP spacing of 3.94 cM. The female and male maps were estimated to 2,336.1 and 1,441.5 cM, respectively. The gene order was validated through comparisons to the cytogenetic and/or physical location of 203 genes, linkage to evenly spaced microsatellite markers as well as previously reported conserved synteny. A total of 330 previously unmapped genes and ESTs were mapped to the porcine autosome while ten genes were mapped to unexpected locations.ConclusionThe linkage map presented here shows high accuracy in gene order. The pedigree family network as well as the large amount of meiotic events provide good reliability and make this map suitable for QTL and association studies. In addition, the linkage to the RH-map of microsatellites makes it suitable for comparison to other QTL studies.

Full-term development of nuclear transfer calves produced from open-pulled straw (OPS) vitrified cytoplasts: Work in progress

Cryopreservation of cytoplasts would help to resolve the logistics of matching the availability of oocytes with embryo donors in nuclear transfer. Therefore, the developmental potential of nuclear transfer bovine embryos reconstructed using vitrified cytoplasts was investigated. In vitro matured oocytes were denuded, enucleated, activated with calcium ionophore (10 microM, 5 min) and cycloheximide (10 microg/mL, 6 h) and then vitrified by the open pulled straw (OPS) method. After immediate warming, the nuclear transfer embryos were reconstructed using blastomeres from nonvitrified,in vitro-produced embryo donors. Compared with control nuclear transfer embryos that were reconstructed using nonvitrified cytoplasts, fusion rates (% +/- SEM) were not affected (83.7+/-9.2 vs. 79.8+/-4.6; P>0.05), but cleavage (55.7+/-2.9 vs. 92.8+/-3.9; P = 0.0002) and blastocyst rates (7.2+/-5.0 vs. 32.6+/-7.8; P = 0.0025, vitrified vs. nonvitrified cytoplasts, respectively) per successful fusion were reduced. One nuclear transfer blastocyst reconstructed from a vitrified cytoplast was transferred to a synchronized recipient. After a normal length gestation (265 d), twin calves (21 and 26 kg) were delivered. Microsatellite analysis confirmed that the calves were homozygotic (the embryo split in utero), and were derived from the in vitro-produced embryo donor. The twins were dead at birth, but post-mortem analysis of the calves indicated no abnormalities or infections, suggesting that their death was related to the twin pregnancy and the known fragility of nuclear transfer calves. These data demonstrate that open pulled straw-vitrified cytoplasts are capable of supporting full-term development of nuclear transfer embryos.

Circulating surfactant protein D is decreased in systemic lupus erythematosus.

Objective. Deficiencies of innate immune molecules like mannan binding lectin (MBL) have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). Surfactant protein D (SP-D) and MBL belong to the same family of innate immune molecules — the collectins, which share important structural and functional properties. We aimed to compare concentrations of serum SP-D in patients with SLE and in healthy controls, and to investigate if SP-D is associated with selected disease indicators. We investigated the possible association of the Met11Thr polymorphism with disease, since this polymorphism is an important determinant for serum level, oligomerization pattern, and function of SP-D. Methods. Serum SP-D was measured using a 5-layer ELISA in 70 SLE patients and 1476 healthy subjects. DNA was genotyped for the Met11Thr variant. Results. Median SP-D level in serum was 911 ng/ml (95% CI 776–1118) in patients and 1068 ng/ml (95% CI 901–1246) in controls (p = 0.0004). Circulating SP-D did not differ significantly in patients with high, intermediate, or low SLE disease activity. Similarly, SP-D did not correlate with C-reactive protein, erythrocyte sedimentation rate, and anti-dsDNA seropositivity. Genetic analysis did not support an association of the Met11Thr genotype with SLE. Conclusion. These findings suggest that low SP-D, unrelated to conventional disease indicators, represents an aspect of SLE etiopathogenesis.