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PeerJ2018; 6; e5759; doi: 10.7717/peerj.5759

Sequence variant analysis of RNA sequences in severe equine asthma.

Abstract: 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 and . Sanger sequencing confirmed that the substitution was appropriately identified in all 26 samples while the 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 , no difference in allele frequency between the two groups was detected but a higher proportion of asthmatic horses had the altered allele compared to non-asthmatic animals. Conclusions: 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.
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Publication Date: 2018-10-11 PubMed ID: 30324028PubMed Central: PMC6186407DOI: 10.7717/peerj.5759Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research aimed to identify genetic variants potentially associated with severe equine asthma using RNA-Seq, a technology that involves analyzing sequences of RNA. This type of asthma in horses is a chronic lung disease similar to a type of late-onset asthma in humans. The study targeted variant sequence identification to shed light on the origins of asthma.

Methodology

  • The study processed RNA-Seq data from lung biopsies of both asthmatic and non-asthmatic horses, both before and after exposure to asthma triggers.
  • The sequences were then matched to the equine genome using a Spliced Transcripts Alignment to Reference software.
  • Read preparation for sequence variant calling was then performed using Picard tools and Genome Analysis Toolkit (GATK).
  • The sequence variants were identified and filtered using GATK and Ensembl Variant Effect Predictor (VEP) tools.
  • Two RNA-Seq predicted sequence variants were further investigated utilizing both PCR and Sanger sequencing.
  • Additional analysis of novel sequence variant selection was conducted with VEP, relying on a score of under 0.01 predicted with Sorting Intolerant from Tolerant software, and considering other factors like missense nature, location within the protein coding sequence, and presence in all asthmatic individuals.
  • The consequences of select variants in protein functionality were 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 of frequency in alleles between the groups was calculated using a Pearson’s Chi-squared test with Yates’ continuity correction. The difference of genotype frequency was calculated using the Fisher’s exact test for count data.

Results

  • The RNA-Seq variant calling and filtering method successfully identified two substitution variants.
  • These sequences were then confirmed useful by Sanger sequencing. Specifically, one variant was established in all samples, and the other was set in 24 out of 26 samples.
  • The identified variants are believed to cause loss of function and not be neutral, expected to impact the development of severe equine asthma.
  • Amino acid substitutions predicted no change for PACRG protein, but a change for RTTN.
  • One of the variants did not display any difference between the two groups, but a higher proportion of asthmatic horses carried the altered allele.

Conclusions

  • RNA-Seq proved both sensitive and specific for calling gene sequence variants in this disease model.
  • The methodology had a 92% correct identification rate even with moderate coverage. This suggests the method’s possible suitability to detect sequence variants in samples with low coverage.
  • Although the effects of the amino acid changes in the PACRG and RTTN proteins are still uncertain, their connection with ciliary function may be important for future research.

Cite This Article

APA
Tessier L, Côté O, Bienzle D. (2018). Sequence variant analysis of RNA sequences in severe equine asthma. PeerJ, 6, e5759. https://doi.org/10.7717/peerj.5759

Publication

PeerJ
ISSN: 2167-8359
NlmUniqueID: 101603425
Country: United States
Language: English
Volume: 6
Pages: e5759
PII: e5759

Researcher Affiliations

Tessier, Laurence
  • Department of Pathobiology, University of Guelph, Guelph, ON, Canada.
  • BenchSci, Toronto, ON, Canada.
Côté, Olivier
  • Department of Pathobiology, University of Guelph, Guelph, ON, Canada.
  • BioAssay Works, Ijamsville, MD, USA.
Bienzle, Dorothee
  • Department of Pathobiology, University of Guelph, Guelph, ON, Canada.

Conflict of Interest Statement

Olivier Côté is employed by BioAssay Works LLC, Ijamsville, MD, USA. Laurence Tessier became an employee of BenchSci, Toronto, ON after completion of this study. Dorothee Bienzle is an Academic Editor for PeerJ.

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