Abstract
ObjectiveThe Arabidopsis thaliana Niederzenz-1 genome sequence was recently published with an ab initio gene prediction. In depth analysis of the predicted gene set revealed some errors involving genes with non-canonical splice sites in their introns. Since non-canonical splice sites are difficult to predict ab initio, we checked for options to improve the annotation by transferring annotation information from the recently released Columbia-0 reference genome sequence annotation Araport11.ResultsIncorporation of hints generated from Araport11 enabled the precise prediction of non-canonical splice sites. Manual inspection of RNA-Seq read mapping and RT-PCR were applied to validate the structural annotations of non-canonical splice sites. Predictions of untranslated regions were also updated by harnessing the potential of Araport11’s information, which was generated by using high coverage RNA-Seq data. The improved gene set of the Nd-1 genome assembly (GeneSet_Nd-1_v1.1) was evaluated via comparison to the initial gene prediction (GeneSet_Nd-1_v1.0) as well as against Araport11 for the Col-0 reference genome sequence. GeneSet_Nd-1_v1.1 contains previously missed non-canonical splice sites in 1256 genes. Reciprocal best hits for 24,527 (89.4%) of all nuclear Col-0 genes against the GeneSet_Nd-1_v1.1 indicate a high gene prediction quality.
Highlights
Eukaryotic genes are transcribed as a primary transcript that is subsequently converted to a mature mRNA through several processing steps including splicing
To correlate and compare gene structures from related genomes, the first step is to define “orthologous” gene couples. Such couples can efficiently be determined by evaluating reciprocal best BLAST hits (RBHs) [32,33,34,35]
Each RBH couple consists of two genes, one from each of the two genome sequences to compare, which display the highest scoring hit in the other data set in a reciprocal manner [36]
Summary
Incorporation of hints generated from Araport enabled the precise prediction of non-canonical splice sites. Manual inspection of RNA-Seq read mapping and RT-PCR were applied to validate the structural annotations of non-canonical splice sites. Predictions of untranslated regions were updated by harnessing the potential of Araport11’s information, which was generated by using high coverage RNA-Seq data. The improved gene set of the Nd-1 genome assembly (GeneSet_Nd-1_v1.1) was evaluated via comparison to the initial gene prediction (GeneSet_Nd-1_v1.0) as well as against Araport for the Col-0 reference genome sequence. GeneSet_Nd-1_v1.1 contains previously missed non-canonical splice sites in 1256 genes. Reciprocal best hits for 24,527 (89.4%) of all nuclear Col-0 genes against the GeneSet_Nd-1_v1.1 indicate a high gene prediction quality
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