What's in your next-generation sequence data? An exploration of unmapped DNA and RNA sequence reads from the bovine reference individual.

BackgroundNext-generation sequencing projects commonly commence by aligning reads to a reference genome assembly. While improvements in alignment algorithms and computational hardware have greatly enhanced the efficiency and accuracy of alignments, a significant percentage of reads often remain unmapped.ResultsWe generated de novo assemblies of unmapped reads from the DNA and RNA sequencing of the Bos taurus reference individual and identified the closest matching sequence to each contig by alignment to the NCBI non-redundant nucleotide database using BLAST. As expected, many of these contigs represent vertebrate sequence that is absent, incomplete, or misassembled in the UMD3.1 reference assembly. However, numerous additional contigs represent invertebrate species. Most prominent were several species of Spirurid nematodes and a blood-borne parasite, Babesia bigemina. These species are either not present in the US or are not known to infect taurine cattle and the reference animal appears to have been host to unsequenced sister species.ConclusionsWe demonstrate the importance of exploring unmapped reads to ascertain sequences that are either absent or misassembled in the reference assembly and for detecting sequences indicative of parasitic or commensal organisms

Prospecção de SNPs no gene TNFa e sua possível associação à verminose gastrintestinal em caprinos.

O objetivo desse estudo foi investigar a associação entre marcadores moleculares do tipo SNP (Polimorfismo de base única) prospectados no gene TNFa (fator de necrose tumoral alfa) e resistência à verminose gastrintestinal em caprinos. Para isso, foram produzidos 229 caprinos a partir de animais das raças Saanen, raça considerada susceptível a endoparasitas gastrintestinais e Anglo-nubiana, raça considerada resistente. Para a prospecção de SNPs, foram sequenciados 44 animais extremos para OPG (contagem de ovos por grama de fezes) escolhidos a partir dos resíduos obtidos através do modelo estatístico usado para corrigir os dados fenotípicos para as variações ambientais. O sequenciamento inclui quatro regiões do gene TNFa. Nestas regiões, foram encontrados dez SNPs: dois estão localizados na região promotora do gene, dois no intron 2 e seis no exon 4. Através da aplicação do teste de Fisher foi encontrada uma associação de cinco dos SNPs prospectados ( P ≤ 0,05), o que indica que esses SNPs são potenciais marcadores moleculares para resistência à verminose gastrintestinal

A network landscape from CNVs to Nellore cattle beef tenderness.

This work aims to perform a couple of in silico network analysis from CNVs standpoint, shedding light to possible metabolic connections to tenderness. It was used 671 Nellore males to infer CNVs, through SNP-chip (Illumina Bovine HD Beadchip®, containing approximately 770 thousand SNPs) and PennCNV software methodology. CNV regions (CNVRs) were inferred by CNVRuler (recurrence 0.1).ISAFG 2013. AB.01

A network landscape from CNVs to Nellore cattle beef tenderness.

This work aims to perform a couple of in silico network analysis from CNVs standpoint, shedding light to possible metabolic connections to tenderness. It was used 671 Nellore males to infer CNVs, through SNP-chip (Illumina Bovine HD Beadchip®, containing approximately 770 thousand SNPs) and PennCNV software methodology. CNV regions (CNVRs) were inferred by CNVRuler (recurrence 0.1).ISAFG 2013. AB.01

Análise haplotípica do gene ASAP1 associado com maciez de carne em bovinos da raça Nelore.

A maciez da carne tem impacto importante na satisfação dos consumidores, portanto, o conhecimento da variação desta característica, assim como a prospecção de genes ou segmentos genômicos que influenciem a mesma, pode ajudar no desenvolvimento de critérios quantitativos e moleculares para seleção de bovinos de corte. A análise haplotípica com os SNPs do gene ASAP1 representados no Illumina BovineHD BeadChip mostram que estes marcadores estão associados com força de cisalhamento medida após 7 dias de maturação da carne nessa população da raça Nelore. A validação desses resultados em outras populações poderá contribuir para inclusão desses marcadores em programas de melhoramento da raça Nelore

Tissue Tropism in Host Transcriptional Response to Members of the Bovine Respiratory Disease Complex.

Bovine respiratory disease (BRD) is the most common infectious disease of beef and dairy cattle and is characterized by a complex infectious etiology that includes a variety of viral and bacterial pathogens. We examined the global changes in mRNA abundance in healthy lung and lung lesions and in the lymphoid tissues bronchial lymph node, retropharyngeal lymph node, nasopharyngeal lymph node and pharyngeal tonsil collected at the peak of clinical disease from beef cattle experimentally challenged with either bovine respiratory syncytial virus, infectious bovine rhinotracheitis, bovine viral diarrhea virus, Mannheimia haemolytica or Mycoplasma bovis. We identified signatures of tissue-specific transcriptional responses indicative of tropism in the coordination of host's immune tissue responses to infection by viral or bacterial infections. Furthermore, our study shows that this tissue tropism in host transcriptional response to BRD pathogens results in the activation of different networks of response genes. The differential crosstalk among genes expressed in lymphoid tissues was predicted to be orchestrated by specific immune genes that act as 'key players' within expression networks. The results of this study serve as a basis for the development of innovative therapeutic strategies and for the selection of cattle with enhanced resistance to BRD

Finding a minimal set of SNPs for paternity identification in Nelore cattle.

Cattle breeding programs often demand pedigree information to associate the herdability of genetic markers to animal performance, like milk and beef quality or disease predisposition. Paternity mistakes are a common problem in cattle herds and genetic markers have been used in parentage exclusion tests for resolving pedigree disagreements. The most common markers used in these tests are microsatellites and SNPs (Single Nucleotide Polymorphisms). SNPs have also been used in GWAS (Genome Wide Association Studies) based on the novel commercial high density bovine SNP chips. Such platforms possess around 770k SNPs which can be used also as a rich resource for paternity exclusion tests. However genotyping such a large amount of SNPs for individuals of a whole flock to perform paternity exclusion tests is not economically feasible. Furthermore, many of these SNPs are non-informative because of high levels of linkage disequilibrium among them and because of low levels of minimum allele frequency (MAF), call rate and genotyping accuracy (GC score). In this work we devised a method to select a minimal set of SNPs from Nelore cattle samples, which are highly informative and are independent, so they could be used in a small genotyping panel for paternity exclusion tests. Samples from 32 Nelore sires chosen to represent the main genealogies marketed in Brazil and 762 nelore bulls produced by artificial insemination were genotyped. The platform used was Illumina BovineHD Genotyping Beadchip that has ~770k SNPs. The algorithm developed first run a quality control filter that selected, from the starting 770k SNPs, only markers that: i) were in Hardy Weinberg equilibrium, ii) had MAF>0.4, iii) had Illumina's accuracy GC Score > 0.8, iv) had call rate >=0.95 and v) had a D?<0.5 with other marker. 126 pairs of father-sons (151 samples), with the paternity already resolved, were selected to compose the test set. The other 643 animals that were left composed the training set. From the training set, 325 SNPs passed the quality control filter. The selected 325 SNPs were used in the test set to perform paternity tests, following the methodology adapted from Fung et al., 2002. The results show that values of the combined power of exclusion (Q) for all markers, between 0.2 and 0.7, gives the correct paternity answer for all pairs of individuals. We believe that this set of 325 SNPs could be used in a small genotyping chip to perform paternity exclusion tests for Nelore cattle; a low-cost solution to be used by cattle producers to correct paternity disagreements. All procedures were performed using the R language in a local linux server machine.X-MEETING, 2011

Prospecção de SNPs no gene TNFa e sua possível associação à verminose gastrintestinal em caprinos.

Resumo: O objetivo desse estudo foi investigar a associação entre marcadores moleculares do tipo SNP (Polimorfismo de base única) prospectados no gene TNFa (fator de necrose tumoral alfa) e resistência à verminose gastrintestinal em caprinos. Para isso, foram produzidos 229 caprinos a partir de animais das raças Saanen, raça considerada susceptível a endoparasitas gastrintestinais e Anglo-nubiana, raça considerada resistente. Para a prospecção de SNPs, foram sequenciados 44 animais extremos para OPG (contagem de ovos por grama de fezes) escolhidos a partir dos resíduos obtidos através do modelo estatístico usado para corrigir os dados fenotípicos para as variações ambientais. O sequenciamento inclui quatro regiões do gene TNFa. Nestas regiões, foram encontrados dez SNPs: dois estão localizados na região promotora do gene, dois no intron 2 e seis no exon 4. Através da aplicação do teste de Fisher foi encontrada uma associação de cinco dos SNPs prospectados ( P ? 0,05), o que indica que esses SNPs são potenciais marcadores moleculares para resistência à verminose gastrintestinal. Prospection of SNPs in TNFa gene and possible association with gastrointestinal endoparasites resistance Abstract: The aim of this study was to investigate the association between prospected molecular markers, SNPs (Single Nucleotide Polymorphism) in the TNFa gene and resistance to gastrointestinal nematode parasites in goats. For this, 229 goats were produced from animals of Saanen, breed considered susceptible to gastrointestinal endoparasites and Anglo-Nubian breed considered resistant. For SNP prospection, 44 extreme animals for fecal egg counting (FEC) chosen residues of a statistical model that corrected the phenotype for environmental variations and, from four regions of the gene TNFa were sequenced. Ten SNPs were found: two are located in the promoter region, two in the intron 2 and six in the exon 4. By applying the Fisher test association of five of the SNPs prospected (P ? 0.05) were found indicating that these SNPs are potential molecular markers for resistance to gastrointestinal nematode parasites

Comparação de diferentes protocolos para visualização de DNA separado por eletroforese em gel de agarose utilizando-se os corantes fluorescentes brometo de etídeo e GelRedr.

O brometo de etídio é um corante fluorescente mutagênico utilizado para a visualização de ácidos nucléicos separados pela técnica analítica de eletroforese em gel. Há um corante alternativo, apresentado comercialmente como GelRedR (Biotium), que tem se destacado por não ter ação mutagênica. Sua estrutura ainda não é de conhecimento público por estar sob processo de patente, porém sabe-se que sua massa molar é pelo menos 3 vezes maior do que a do primeiro. Comumente, o brometo de etídeo, e mais recentemente o GelRed são utilizados segundo diferentes protocolos, utilizados no gel de agarose antes da polimerização do mesmo, em banho, após a eletroforese ou diretamente na amostra