Gene Expression Networks in the Drosophila Genetic Reference Panel

A major challenge of modern biology is to understand how naturally occurring variation in DNA sequences affects complex organismal phenotypes through networks of intermediate molecular phenotypes. Here, we performed deep RNA sequencing of 200 Drosophila Genetic Reference Panel inbred lines with complete genome sequences and mapped expression quantitative trait loci for annotated genes, novel transcribed regions (most of which are long noncoding RNAs), transposable elements and microbial species. We identified host variants affecting expression of transposable elements independent of copy number and microbiome composition. We constructed sex-specific expression quantitative trait locus regulatory networks. These networks are enriched for novel transcribed regions and target genes in heterochromatin and euchromatic regions of reduced recombination and are associated with transposable element expression. This study uncovers novel genetic features that regulate natural genetic variation of gene expression and generates testable hypotheses for future functional analyses. Overall design: We performed deep RNA sequencing of the Drosophila melanogaster Genetic Reference Panel (DGRP) of inbred lines with complete DNA sequences. We produced rRNA-depleted strand-specific total RNA-seq libraries for 200 DGRP lines. For each line, we collected two replicate pools of 3-5 day old mated flies, with 25 females and 30 males per line. cDNA libraries were prepared separately for each replicate and each sex, for a total of 800 RNA-seq samples (200 lines x 2 sexes x 2 replicates). All flies were reared in identical conditions and collected at the same time of day, using a strict randomized experimental design for sample collection.