Mated females were collected from a population in Raleigh, North Carolina, USA; the progeny of each female were inbred via full-sibling matings for 20 generations. Genomic sequence of 168 of the inbred lines was determined using Illumina and/or 454 sequencing technology; sequence reads were mapped to the D. melanogaster reference genome (iso-1 strain).

4,672,297 single nucleotide polymorphisms (SNPs) were called using the Joint Genotyper for Inbred Lines (JGIL), which takes into account coverage and quality sequencing statistics, and expected allele frequencies after 20 generations of inbreeding from an outbred population initially in Hardy–Weinberg equilibrium. In cases where base calls were made by both technologies, concordance was 99.36%.