Re‐replication Origins in Sciara DNA Puffs Revealed by New and Old Genomic Technologies including Nanopore Sequencing

Abstract

Each one of the numerous origins of DNA replication (ORIs) in metazoan genomes is typically regulated to fire no more than once in any given S‐phase. “Intrachromosomal DNA amplification” occurs when an ORI re‐fires leading to extra copies of that locus. This event is rare, aberrant, and deadly in most metazoans, which makes it difficult to study. However, the fungus fly Sciara coprophila has several “DNA puffs” in the salivary gland genome where multiple rounds of DNA re‐replication occur during normal larval development, offering a unique opportunity to investigate how particular ORIs can re‐fire while the other ORIs in the genome do not. Previously, we studied the amplification ORI at locus II/9A and showed that a steroid hormone, ecdysone, plays a role in its repeated firing. Expanding our studies to the other DNA puffs is a first step in further understanding the mechanism of DNA re‐replication in Sciara. However, the other DNA puff sequences were unknown as was the Sciara genome sequence. To obtain new DNA puff sequences we first assembled the genome using many genomics technologies, including Illumina, PacBio, BioNano Genomics Irys, and ultra‐long reads from Oxford Nanopore minION. With the assembled genome we used Illumina sequencing of salivary gland genomes before, during and after amplification followed by copy number analysis to identify new DNA puffs. In addition, we have sequenced the salivary transcriptomes before, during, and after amplification to search for candidate genes that are involved in regulating DNA re‐replication in Sciara. [JMU supported by NSF GRFP (DGE‐1058262) NSF EPSCoR (grant# 1004057) fellowships]