Variant analysis of RNA sequences in severe equine asthma
- Published
- Accepted
- Subject Areas
- Genetics, Veterinary Medicine, Allergy and Clinical Immunology, Respiratory Medicine
- Keywords
- Severe asthma, recurrent airway obstruction, equine, single nucleotide variant, cilia, rotatin, Parkin co-regulated, RTTN, PACRG
- Copyright
- © 2018 Tessier et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2018. Variant analysis of RNA sequences in severe equine asthma. PeerJ Preprints 6:e3491v2 https://doi.org/10.7287/peerj.preprints.3491v2
Abstract
Background. Severe 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 or potential relevance to the pathogenesis of asthma. Methods. RNA-Seq data were generated from endobronchial biopsies collected from 6 asthmatic and 7 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 (SIFT) 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 (PolyPhen) 2 and Screening for Non-Acceptable Polymorphism (SNAP) 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. Results. RNA-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. Discussion. RNA-Seq was sensitive and specific for calling gene sequence variants in this disease model. Even moderate coverage (<10-20 cpm) 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.
Author Comment
This version has greater emphasis on the methodological innovation and lesser emphasis on the particular variants identified.
Supplemental Information
Variants detected in asthmatic and non-asthmatic horses with Genome Analysis Toolkit (GATK)
The Venn diagram was generated with SeqMule to identify variants present in all asthmatics before (red) and after challenge (green), and in non-asthmatics before (turquoise) and after challenge (purple).
Predicted effect of variants in all (left) and coding (right) regions for asthmatic horses before (upper) and after (lower) challenge
Analysis was done using Ensembl Variant Effect Predictor (VEP) tools.
Predicted effect of variants from all (left) and coding (right) regions for asthmatic horses before (upper) and after (lower) challenge
Analysis was done using Ensembl Variant Effect Predictor (VEP) tools.
Results of analysis of PACRG (A) and RTTN (B) mutations with PolyPhen2 software
Mutations were predicted as probably damaging in PACRG and RTTN proteins with confidence scores of 0.993 and 0.979, respectively.