Substantial differences in bias between single‐digest and double‐digest RAD‐seq libraries: A case study

The trade‐offs of using single‐digest vs. double‐digest restriction site‐associated DNA sequencing (RAD‐seq) protocols have been widely discussed. However, no direct empirical comparisons of the two methods have been conducted. Here, we sampled a single population of Gulf pipefish (Syngnathus scovelli) and genotyped 444 individuals using RAD‐seq. Sixty individuals were subjected to single‐digest RAD‐seq (sdRAD‐seq), and the remaining 384 individuals were genotyped using a double‐digest RAD‐seq (ddRAD‐seq) protocol. We analysed the resulting Illumina sequencing data and compared the two genotyping methods when reads were analysed either together or separately. Coverage statistics, observed heterozygosity, and allele frequencies differed significantly between the two protocols, as did the results of selection components analysis. We also performed an in silico digestion of the Gulf pipefish genome and modelled five major sources of bias: PCR duplicates, polymorphic restriction sites, shearing bias, asymmetric sampling (i.e., genotyping fewer individuals with sdRAD‐seq than with ddRAD‐seq) and higher major allele frequencies. This combination of approaches allowed us to determine that polymorphic restriction sites, an asymmetric sampling scheme, mean allele frequencies and to some extent PCR duplicates all contribute to different estimates of allele frequencies between samples genotyped using sdRAD‐seq versus ddRAD‐seq. Our finding that sdRAD‐seq and ddRAD‐seq can result in different allele frequencies has implications for comparisons across studies and techniques that endeavour to identify genomewide signatures of evolutionary processes in natural populations.     

Adaptable gene‐specific dye bias correction for two‐channel DNA microarrays

DNA microarray technology is a powerful tool for monitoring gene expression or for finding the location of DNA‐bound proteins. DNA microarrays can suffer from gene‐specific dye bias (GSDB), causing some probes to be affected more by the dye than by the sample. This results in large measurement errors, which vary considerably for different probes and also across different hybridizations. GSDB is not corrected by conventional normalization and has been difficult to address systematically because of its variance. We show that GSDB is influenced by label incorporation efficiency, explaining the variation of GSDB across different hybridizations. A correction method (Gene‐ And Slide‐Specific Correction, GASSCO) is presented, whereby sequence‐specific corrections are modulated by the overall bias of individual hybridizations. GASSCO outperforms earlier methods and works well on a variety of publically available datasets covering a range of platforms, organisms and applications, including ChIP on chip. A sequence‐based model is also presented, which predicts which probes will suffer most from GSDB, useful for microarray probe design and correction of individual hybridizations. Software implementing the method is publicly available.     

A gene silencing of V‐ATPase subunit A interferes with survival and development of the tomato leafminer,Tuta absoluta

RNA interference (RNAi) technology is not only considered as a tool to analyze gene function, but it is also potentially considered as a strategy to develop novel biopesticide. In the current study, a double‐stranded RNA specific to v‐ATPase subunit A of the tomato leafminer, Tuta absoluta (Meyrick; Lepidoptera: Gelechiidae), was orally administered. A gradual decrease in the expression of the gene was observed from Day 1 to 3 and resulted in significant larval mortality. These results suggest that v‐ATPases A can be considered as a promising target gene by RNAi technology to be used in the management of the tomato leafminer.     

RNA‐sequencing profiles hippocampal gene expression in a validated model of cancer‐induced depression

To investigate the pathophysiology of cancer‐induced depression (CID), we have recently developed a validated CID mouse model. Given that the efficacy of antidepressants in cancer patients is controversial, it remains unclear whether CID is a biologically distinct form of depression. We used RNA‐sequencing (RNA‐seq) to investigate differentially expressed genes (DEGs) in hippocampi of animals from our CID model relative a positive control model of depressive‐like behavior induced with chronic corticosterone (CORT). To validate RNA‐seq results, we performed quantitative real‐time RT‐PCR (qRT‐PCR) on a subset of DEGs. Enrichment analysis using DAVID was performed on DEGs to identify enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and biological process gene ontologies (GO:BP). qRT‐PCR results significantly predicted RNA‐seq results. RNA‐seq revealed that most DEGs identified in the CORT model overlapped with the CID model. Enrichment analyses identified KEGG pathways and GO:BP terms associated with ion homeostasis and neuronal communication for both the CORT and CID model. In addition, CID DEGs were enriched in pathways and terms relating to neuronal development, intracellular signaling, learning and memory. This study is the first to investigate CID at the mRNA level. We have shown that most hippocampal mRNA changes that are associated with a depressive‐like state are also associated with cancer. Several other changes occur at the mRNA level in cancer, suggesting that the CID model may represent a biologically distinct form of a depressive‐like state.     

Complex patterns of cis‐regulatory polymorphisms in ebony underlie standing pigmentation variation in Drosophila melanogaster

Pigmentation traits in adult Drosophila melanogaster were used in this study to investigate how phenotypic variations in continuous ecological traits can be maintained in a natural population. First, pigmentation variation in the adult female was measured at seven different body positions in 20 strains from the Drosophila melanogaster Genetic Reference Panel (DGRP) originating from a natural population in North Carolina. Next, to assess the contributions of cis‐regulatory polymorphisms of the genes involved in the melanin biosynthesis pathway, allele‐specific expression levels of four genes were quantified by amplicon sequencing using a 454 GS Junior. Among those genes, ebony was significantly associated with pigmentation intensity of the thoracic segment. Detailed sequence analysis of the gene regulatory regions of this gene indicated that many different functional cis‐regulatory alleles are segregating in the population and that variations outside the core enhancer element could potentially play important roles in the regulation of gene expression. In addition, a slight enrichment of distantly associated SNP pairs was observed in the ~10 kb cis‐regulatory region of ebony, which suggested the presence of interacting elements scattered across the region. In contrast, sequence analysis in the core cis‐regulatory region of tan indicated that SNPs within the region are significantly associated with allele‐specific expression level of this gene. Collectively, the data suggest that the underlying genetic differences in the cis‐regulatory regions that control intraspecific pigmentation variation can be more complex than those of interspecific pigmentation trait differences, where causal genetic changes are typically confined to modular enhancer elements.