Sample richness and genetic diversity as drivers of chimera formation in nSSU metagenetic analyses

Eukaryotic diversity in environmental samples is often assessed via PCR-based amplification of nSSU genes. However, estimates of diversity derived from pyrosequencing environmental data sets are often inflated, mainly because of the formation of chimeric sequences during PCR amplification. Chimeras are hybrid products composed of distinct parental sequences that can lead to the misinterpretation of diversity estimates. We have analyzed the effect of sample richness, evenness and phylogenetic diversity on the formation of chimeras using a nSSU data set derived from 454 Roche pyrosequencing of replicated, large control pools of closely and distantly related nematode mock communities, of known intragenomic identity and richness. To further investigate how chimeric molecules are formed, the nSSU gene secondary structure was analyzed in several individuals. For the first time in eukaryotes, chimera formation proved to be higher in both richer and more genetically diverse samples, thus providing a novel perspective of chimera formation in pyrosequenced environmental data sets. Findings contribute to a better understanding of the nature and mechanisms involved in chimera formation during PCR amplification of environmentally derived DNA. Moreover, given the similarities between biodiversity analyses using amplicon sequencing and those used to assess genomic variation, our findings have potential broad application for identifying genetic variation in homologous loci or multigene families in general.     

Robust Prediction of Expression Differences among Human Individuals Using Only Genotype Information

Many genetic variants that are significantly correlated to gene expression changes across human individuals have been identified, but the ability of these variants to predict expression of unseen individuals has rarely been evaluated. Here, we devise an algorithm that, given training expression and genotype data for a set of individuals, predicts the expression of genes of unseen test individuals given only their genotype in the local genomic vicinity of the predicted gene. Notably, the resulting predictions are remarkably robust in that they agree well between the training and test sets, even when the training and test sets consist of individuals from distinct populations. Thus, although the overall number of genes that can be predicted is relatively small, as expected from our choice to ignore effects such as environmental factors and trans sequence variation, the robust nature of the predictions means that the identity and quantitative degree to which genes can be predicted is known in advance. We also present an extension that incorporates heterogeneous types of genomic annotations to differentially weigh the importance of the various genetic variants, and we show that assigning higher weights to variants with particular annotations such as proximity to genes and high regional G/C content can further improve the predictions. Finally, genes that are successfully predicted have, on average, higher expression and more variability across individuals, providing insight into the characteristics of the types of genes that can be predicted from their cis genetic variation.     

Chimeric RNAs as potential biomarkers for tumor diagnosis

Cancers claim millions of lives each year. Early detection that can enable a higher chance of cure is of paramount importance to cancer patients. However, diagnostic tools for many forms of tumors have been lacking. Over the last few years, studies of chimeric RNAs as biomarkers have emerged. Numerous reports using bioinformatics and screening methodologies have described more than 30,000 expressed sequence tags (EST) or cDNA sequences as putative chimeric RNAs. While cancer cells have been well known to contain fusion genes derived from chromosomal translocations, rearrangements or deletions, recent studies suggest that trans-splicing in cells may be another source of chimeric RNA production. Unlike cis-splicing, trans-splicing takes place between two pre-mRNA molecules, which are in most cases derived from two different genes, generating a chimeric non-co-linear RNA. It is possible that trans-splicing occurs in normal cells at high frequencies but the resulting chimeric RNAs exist only at low levels. However the levels of certain RNA chimeras may be elevated in cancers, leading to the formation of fusion genes. In light of the fact that chimeric RNAs have been shown to be overrepresented in various tumors, studies of the mechanisms that produce chimeric RNAs and identification of signature RNA chimeras as biomarkers present an opportunity for the development of diagnoses for early tumor detection. [BMB reports 2012; 45(3): 133-140].     

Formation and Longevity of Chimeric and Duplicate Genes in Drosophila melanogaster

Historically, duplicate genes have been regarded as a major source of novel genetic material. However, recent work suggests that chimeric genes formed through the fusion of pieces of different genes may also contribute to the evolution of novel functions. To compare the contribution of chimeric and duplicate genes to genome evolution, we measured their prevalence and persistence within Drosophila melanogaster. We find that ~80.4 duplicates form per million years, but most are rapidly eliminated from the genome, leaving only 4.1% to be preserved by natural selection. Chimeras form at a comparatively modest rate of ~11.4 per million years but follow a similar pattern of decay, with ultimately only 1.4% of chimeras preserved. We propose two mechanisms of chimeric gene formation, which rely entirely on local, DNA-based mutations to explain the structure and placement of the youngest chimeric genes observed. One involves imprecise excision of an unpaired duplication during large-loop mismatch repair, while the other invokes a process akin to replication slippage to form a chimeric gene in a single event. Our results paint a dynamic picture of both chimeras and duplicate genes within the genome and suggest that chimeric genes contribute substantially to genomic novelty.