ONT-Based Alternative Assemblies Impact on the Annotations of Unique versus Repetitive Features in the Genome of a Romanian Strain of Drosophila melanogaster

Abstract

To date, different strategies of whole-genome sequencing (WGS) have been developed in order to understand the genome structure and functions. However, the analysis of genomic sequences obtained from natural populations is challenging and the biological interpretation of sequencing data remains the main issue. The MinION device developed by Oxford Nanopore Technologies (ONT) is able to generate long reads with minimal costs and time requirements. These valuable assets qualify it as a suitable method for performing WGS, especially in small laboratories. The long reads resulted using this sequencing approach can cover large structural variants and repetitive sequences commonly present in the genomes of eukaryotes. Using MinION, we performed two WGS assessments of a Romanian local strain of Drosophila melanogaster, referred to as Horezu_LaPeri (Horezu). In total, 1,317,857 reads with a size of 8.9 gigabytes (Gb) were generated. Canu and Flye de novo assembly tools were employed to obtain four distinct assemblies with both unfiltered and filtered reads, achieving maximum reference genome coverages of 94.8% (Canu) and 91.4% (Flye). In order to test the quality of these assemblies, we performed a two-step evaluation. Firstly, we considered the BUSCO scores and inquired for a supplemental set of genes using BLAST. Subsequently, we appraised the total content of natural transposons (NTs) relative to the reference genome (ISO1 strain) and mapped the mdg1 retroelement as a resolution assayer. Our results reveal that filtered data provide only slightly enhanced results when considering genes identification, but the use of unfiltered data had a consistent positive impact on the global evaluation of the NTs content. Our comparative studies also revealed differences between Flye and Canu assemblies regarding the annotation of unique versus repetitive genomic features. In our hands, Flye proved to be moderately better for gene identification, while Canu clearly outperformed Flye for NTs analysis. Data concerning the NTs content were compared to those obtained with ONT for the D. melanogaster ISO1 strain, revealing that our strategy conducted better results. Additionally, the parameters of our ONT reads and assemblies are similar to those reported for ONT experiments performed on various model organisms, revealing that our assembly data are appropriate for a proficient annotation of the Horezu genome.

The Landscape of the DNA Transposons in the Genome of the Horezu_LaPeri Strain of Drosophila melanogaster

“…Only the insertions with TSDs ( Supplementary Table S3 ) were PCR checked, and the obtained amplicons were migrated in agarose gel ( Supplementary Figure S1 ). Some of the tested insertions were identified using the Canu assembly analyzed in this paper, while three other P-element insertions (in Ac13E , in retn and near stv ) with identical TSDs were identified in alternative assemblies described elsewhere [ 21 , 24 ]. The PCR primers are specific to the sequences that flank the insertions of the P elements ( Supplementary Table S1 ) and together with the MM11 primer (specific for TIRs of the P-element) were used to amplify the genome–transposon junction, thus testing the presence of the insertions.…”

“…Sequencing data used in this study were obtained from a Romanian local natural strain of D. melanogaster , named Horezu_LaPeri and collected from the location Romanii de Sus, Horezu, Vâlcea County, Romania, in August 2018. Nanopore sequencing of the Horezu_LaPeri genome was performed with the MinION device from ONT and was previously described in detail elsewhere [ 24 ], along with the qualitative parameters of four alternative assemblies generated with Canu v2.1.1 [ 25 ] and Flye v2.8.3 [ 26 ] applications. In our hands, the most reliable assembly for transposon analysis was Canu-Data set I, obtained using unfiltered long reads.…”