Sequence variant analysis of RNA sequences in severe equine asthma

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 of potential relevance to the pathogenesis of...

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Main Authors: Laurence Tessier, Olivier Côté, Dorothee Bienzle
Format: Article
Language:English
Published: PeerJ Inc. 2018-10-01
Series:PeerJ
Subjects:
Online Access:https://peerj.com/articles/5759.pdf
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spelling doaj-f10ba06ffe4f44c5af16dfe9f4c7edb72020-11-24T21:14:32ZengPeerJ Inc.PeerJ2167-83592018-10-016e575910.7717/peerj.5759Sequence variant analysis of RNA sequences in severe equine asthmaLaurence Tessier0Olivier Côté1Dorothee Bienzle2Department of Pathobiology, University of Guelph, Guelph, ON, CanadaDepartment of Pathobiology, University of Guelph, Guelph, ON, CanadaDepartment of Pathobiology, University of Guelph, Guelph, ON, CanadaBackground 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 of potential relevance to the pathogenesis of asthma. Methods RNA-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. 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 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.https://peerj.com/articles/5759.pdfSevere asthmaRecurrent airway obstructionEquineSingle nucleotide variantCiliaRotatin
collection DOAJ
language English
format Article
sources DOAJ
author Laurence Tessier
Olivier Côté
Dorothee Bienzle
spellingShingle Laurence Tessier
Olivier Côté
Dorothee Bienzle
Sequence variant analysis of RNA sequences in severe equine asthma
PeerJ
Severe asthma
Recurrent airway obstruction
Equine
Single nucleotide variant
Cilia
Rotatin
author_facet Laurence Tessier
Olivier Côté
Dorothee Bienzle
author_sort Laurence Tessier
title Sequence variant analysis of RNA sequences in severe equine asthma
title_short Sequence variant analysis of RNA sequences in severe equine asthma
title_full Sequence variant analysis of RNA sequences in severe equine asthma
title_fullStr Sequence variant analysis of RNA sequences in severe equine asthma
title_full_unstemmed Sequence variant analysis of RNA sequences in severe equine asthma
title_sort sequence variant analysis of rna sequences in severe equine asthma
publisher PeerJ Inc.
series PeerJ
issn 2167-8359
publishDate 2018-10-01
description 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 of potential relevance to the pathogenesis of asthma. Methods RNA-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. 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 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.
topic Severe asthma
Recurrent airway obstruction
Equine
Single nucleotide variant
Cilia
Rotatin
url https://peerj.com/articles/5759.pdf
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