Computational protocol: A Mutation in LTBP2 Causes Congenital Glaucoma in Domestic Cats (Felis catus)

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Protocol publication

[…] Total RNA was extracted from the iridocorneal angle and ciliary body, including the adjacent sclera and trabecular meshwork of an affected 2 month-old cat and an unrelated, age-matched healthy control. After confirmation of RNA integrity both samples were transcriptionally profiled using the HiSeq sequencing system (Illumina, San Diego, CA, http://www.illumina.com). The resulting sequence reads were aligned to Felis catus genome build felCat5 using Tophat software (ver. 2.0.9 http://ccb.jhu.edu/software/tophat/index.shtml) []. Run parameters were: Tophat (ver. 2.0.9) (—max-insertion-length 4,—read-mismatches 2,—read-gap-length 4,—read-edit-dist 4, -G with Ensembl gene predictions)Transcript abundance and differential expression were calculated using the software Cufflinks (ver. 2.1.1; http://cufflinks.cbcb.umd.edu/)[], and cummeRbund (ver. 2.4.1; http://compbio.mit.edu/cummeRbund/)[]. Analysis parameters for Cufflinks were (—max-bundle-frags 20000000).To generate cDNA sequences, exon coordinates for each isoform identified were exported from cummeRbund. Consensus variants within these regions were called using samtools mpileup (ver. 0.1.18) and bcftools (ver. 0.1.17-dev)[]. This produces a single variant call at each position based on the most common allele observed in the RNA-Seq reads. Variants with QUAL value ≥ 100 and exon coordinates were passed on to GATK FastaAlternateReferenceMaker (ver. 2.5-2-gf57256b) to produce mRNA sequences. Short regions that remained unresolved in felCat5 were bridged by manual incremental alignments of unmapped sequence reads to the obtained consensus sequence.cDNA sequence translations to amino acid sequences were carried out using ExPASy (http://www.expasy.org) tools and protein domains were mapped using the NCBI conserved domains server (http://www.ncbi.nlm.nih.gov/cdd/). Sequences with substantial open reading frames were aligned using the Clustal Omega implementation at Uniprot (http://www.ebi.ac.uk/Tools/msa/clustalo/)[]. […]

Pipeline specifications

Software tools TopHat, Cufflinks, CummeRbund, SAMtools, bcftools, GATK, Clustal Omega
Databases ExPASy
Applications RNA-seq analysis, Transcriptome data visualization
Organisms Felis catus, Homo sapiens, Rattus norvegicus
Diseases Ectopia Lentis, Glaucoma, Ocular Hypertension, Optic Nerve Diseases