A paper titled - Deep Evolutionary Comparison of Gene Expression Identifies Parallel Recruitment of Trans-Factors in Two Independent Origins of C4 Photosy nthesis came out in PLOS Genetics. The reason it caught our attention is the following section. Orthology assignment is a major problem in RNAseq experiments and the readers may find the method in the paper useful.
To perform comparative analyses of gene expression between C. gynandra and maize it is necessary to be able to identify homologous genes between the species in the absence of a reference genome for C. gynandra. This is non- trivial due to the inherent properties and artefacts of de novo assembled transcriptomes. For example, it is to be expected that following de novo assemblies of RNAseq data, most gene loci will be represented by multiple assembled transcript variants . These transcripts may differ from each other in several ways, for example through single nucleotide polymorphisms, alternative splicing of internal exons, alternative terminal exons and incomplete/chimeric assembly due to low sequence coverage or assembly errors. Homologous transcript identification is further complicated by the large phylogenetic distance between the species being compared. Increased phylogenetic distance leads to a concomitant increase in global sequence divergence between homologous genes in different species. Therefore any method which is specifically designed for assignment of homologues in de novo assembled transcriptomes should be able to identify and group multiple different transcript variants for any given gene to enable comparative analysis of gene expression.
To determine the suitability of existing assignment methods for identifying homologous transcript groups in de novo assembled transcriptomes we used RNAseq data from rice. Here we carried out de novo assembly of the short read data, and computed an abundance estimate for each de novo assembled transcript. We also computed an abundance estimate for each gene locus in the rice reference genome using the same short read data. Several different strategies for identifying homologous transcripts between the de novo assembled transcriptome and the rice reference genome were tested and the accuracy of each strategy was assessed by the global correlation of the abundance estimates that resulted from the assembled transcripts-to-reference- gene homology map. Global correlation is negatively affected both by false positive errors (incorrect homology assignment), false negative errors (missing orthology assignment) and assembly artefacts (partial and chimeric transcripts) and so it is a good measure of the utility of an orthology assignment method for quantitative transcriptome comparisons. When using simple methods such as a Reciprocal Best-BLAST (RBB) or fixed e-value cut-offs for assignment abundance estimate accuracies were low and unsuitable for comparative gene expression analyses (Figure 2A & 2B). Using more complex methods such as OrthoMCL improved abundance estimate accuracy (Figure 2B). However accuracy is still low for comparative analyses of gene expression.
The abundance estimate accuracy tests revealed that there was room for substantial improvement of orthology assignment from de novo assembled transcriptomes. As there are no specific methods currently available which are designed to account for the properties and artefacts of de novo assembled transcriptomes as outlined above, we developed a novel orthology assignment method to facilitate accurate multispecies comparisons of gene expression from de novo transcriptome assemblies. The method uses machine learning to define sequence similarity parameters for gene homologues and thus compensates for the properties and artefacts of de novo assembled transcriptomes. The first step in this method is to undertake a pairwise reciprocal best-BLAST (RBB) analysis (Figure 2C) using the full set of de novo assembled transcripts against a reference set derived from a reference genome. The RBB hits between these two datasets are identified (Figure 2D) and grouped according to the length of the assembled transcript. For each length group the RBB hits are ranked and the e-value of a chosen percentile is recorded. A matrix of all e-values and query sequence lengths is then fit to a quadratic polynomial model by least-squares fitting (Figure 2E). While the RBBs are accepted as homologues, the function describing this curve is used to classify non-RBB transcripts of any given length, those above the curve are assigned as homologues and those below the curve are rejected (Figure 2F). Thus homologue assignment is conditioned on both the assembled transcript length and also the global sequence divergence between the de novo assembled and reference transcriptome. This approach significantly increased the accuracy of abundance estimates derived from de novo assembled transcripts when compared with estimates derived from the genome (Figure 2B and 2G). This accuracy is also robust to large phylogenetic distances. Even when homologous transcripts were identified using an intermediary reference genome (Arabidopsis thaliana), the accuracy of mRNA abundance estimates remained high (Figure 2H). We conclude our assignment method, conditioned on both sequence length and global sequence divergence, is suitable for comparative analyses of gene expression after de novo transcript assembly from short read sequencing. For a detailed description and validation of this method see Text S1. This approach is also suitable for identifying homologous groups in distantly-related species (see Text S1 for validation on Oryza sativa versus A. thaliana). Thus we used this method to enable comparison of gene expression between Cleome gynandra and maize, an equivalent phylogenetic distance. An online implementation of the method is provided for use at www.bioinformatics.plants.ox.ac.uk/annot?ate/index.html.