A vast amount of DNA variation is being identified by increasingly large-scale exome and genome sequencing projects. To be useful, variants require accurate functional annotation and a wide range of tools are available to this end.
McCarthy et al recently demonstrated the large differences in prediction of loss-of-function (LoF) variation when RefSeq and Ensembl transcripts are used for annotation, highlighting the importance of the reference transcripts on which variant functional annotation is based.
A team led by researchers from the Wellcome Trust Sanger Institute have performed a detailed analysis of the similarities and differences between the gene and transcript annotation in the GENCODE and RefSeq genesets. They demonstrate that the GENCODE Comprehensive set is richer in alternative splicing, novel CDSs, novel exons and has higher genomic coverage than RefSeq, while the GENCODE Basic set is very similar to RefSeq.
Using RNAseq data the researchers show that exons and introns unique to one geneset are expressed at a similar level to those common to both. They present evidence that the differences in gene annotation lead to large differences in variant annotation where GENCODE and RefSeq are used as reference transcripts, although this is predominantly confined to non-coding transcripts and UTR sequence, with at most ~30% of LoF variants annotated discordantly.
The researchers also describe an investigation of dominant transcript expression, showing that it both supports the utility of the GENCODE Basic set in providing a smaller set of more highly expressed transcripts and provides a useful, biologically-relevant filter for further reducing the complexity of the transcriptome.
Comparison of GENCODE and RefSeq Annotation in the UCSC genome browser.
The research team concluded that the reference transcripts selected for variant functional annotation do have a large effect on the outcome. The GENCODE Comprehensive transcripts contain more exons, have greater genomic coverage and capture many more variants than RefSeq in both genome and exome datasets, while the GENCODE Basic set shows a higher degree of concordance with RefSeq and has fewer unique features.