Sequence variant analysis of RNA sequences in severe equine asthma

Abstract

BackgroundSevere equine asthma is a chronic inflammatory disease of the lung in horses similar to low-Th2 late-onset asthma in humans. This study aimed to determine the utility of RNA-Seq to call gene sequence variants, and to identify sequence variants of potential relevance to the pathogenesis of asthma.MethodsRNA-Seq data were generated from endobronchial biopsies collected from six asthmatic and seven non-asthmatic horses before and after challenge (26 samples total). Sequences were aligned to the equine genome with Spliced Transcripts Alignment to Reference software. Read preparation for sequence variant calling was performed with Picard tools and Genome Analysis Toolkit (GATK). Sequence variants were called and filtered using GATK and Ensembl Variant Effect Predictor (VEP) tools, and two RNA-Seq predicted sequence variants were investigated with both PCR and Sanger sequencing. Supplementary analysis of novel sequence variant selection with VEP was based on a score of <0.01 predicted with Sorting Intolerant from Tolerant software, missense nature, location within the protein coding sequence and presence in all asthmatic individuals. For select variants, effect on protein function was assessed with Polymorphism Phenotyping 2 and screening for non-acceptable polymorphism 2 software. Sequences were aligned and 3D protein structures predicted with Geneious software. Difference in allele frequency between the groups was assessed using a Pearson’s Chi-squared test with Yates’ continuity correction, and difference in genotype frequency was calculated using the Fisher’s exact test for count data.ResultsRNA-Seq variant calling and filtering correctly identified substitution variants in PACRG and RTTN. Sanger sequencing confirmed that the PACRG substitution was appropriately identified in all 26 samples while the RTTN substitution was identified correctly in 24 of 26 samples. These variants of uncertain significance had substitutions that were predicted to result in loss of function and to be non-neutral. Amino acid substitutions projected no change of hydrophobicity and isoelectric point in PACRG, and a change in both for RTTN. For PACRG, no difference in allele frequency between the two groups was detected but a higher proportion of asthmatic horses had the altered RTTN allele compared to non-asthmatic animals.DiscussionRNA-Seq was sensitive and specific for calling gene sequence variants in this disease model. Even moderate coverage (<10–20 counts per million) yielded correct identification in 92% of samples, suggesting RNA-Seq may be suitable to detect sequence variants in low coverage samples. The impact of amino acid alterations in PACRG and RTTN proteins, and possible association of the sequence variants with asthma, is of uncertain significance, but their role in ciliary function may be of future interest.

Flow cytometric analysis of equine bronchoalveolar lavage fluid cells in horses with and without severe equine asthma

“…The 8 horses used for the flow cytometry experiments were selected from a research herd composed of horses with normal respiratory function, and SEA-susceptible horses. 45 These horses are fed from large hay bale feeders, and had routine vaccinations and anthelmintic treatments. All procedures were approved by the University of Guelph Animal Care Committee (Animal Use Protocols #4185 and #3816) and followed guidelines of the Canadian Council on Animal Care.…”

Single-cell transcriptomics delineates the immune cell landscape in equine lower airways and reveals upregulation of FKBP5 in horses with asthma

“…High throughput RNA sequencing can be valuable to obtain more detailed information based on gene expression and have been used in several studies to study gene expression differences in BALF cells and bronchial biopsies sampled from horses diagnosed with asthma 9,[23][24][25][26] . Traditional bulk RNA-sequencing technologies obtain measurements from a mixture of cells in which intercellular differences are averaged.…”

Improvement in bioconversion efficiency and reduction of ammonia emission by introduction of fruit fermentation broth in a black soldier fly larvae and kitchen waste conversion system

“…Raw sequencing data were denoised by using the exact sequences variant (Tessier et al. , 2018). The chimeric sequences were removed using USEARCH based on the SILVA database (Edgar, 2010).…”