Novel quality metrics allow identifying and generating high‐quality assemblies of piRNA clusters

Abstract

In most animals, it is thought that the proliferation of a transposable element (TE) is stopped when the TE jumps into a piRNA cluster. Despite this central importance, little is known about the composition and the evolutionary dynamics of piRNA clusters. This is largely because piRNA clusters are notoriously difficult to assemble as they are frequently composed of highly repetitive DNA. With long reads, we may finally be able to obtain reliable assemblies of piRNA clusters. Unfortunately, it is unclear how to generate and identify the best assemblies, as many assembly strategies exist and standard quality metrics are ignorant of TEs. To address these problems, we introduce several novel quality metrics that assess: (a) the fraction of completely assembled piRNA clusters, (b) the quality of the assembled clusters and (c) whether an assembly captures the overall TE landscape of an organisms (i.e. the abundance, the number of SNPs and internal deletions of all TE families). The requirements for computing these metrics vary, ranging from annotations of piRNA clusters to consensus sequences of TEs and genomic sequencing data. Using these novel metrics, we evaluate the effect of assembly algorithm, polishing, read length, coverage, residual polymorphisms and finally identify strategies that yield reliable assemblies of piRNA clusters. Based on an optimized approach, we provide assemblies for the two Drosophila melanogaster strains Canton‐S and Pi2. About 80% of known piRNA clusters were assembled in both strains. Finally, we demonstrate the generality of our approach by extending our metrics to humans and Arabidopsis thaliana.

Population-scale long-read sequencing uncovers transposable elements associated with gene expression variation and adaptive signatures in Drosophila

“…Genome assembly, polishing, deduplication and contaminant removal resulted in genomes with a number of contigs ranging from 153 to 1185 (average 367), genome sizes from 136.6 Mb to 151.3 Mb (average 142 Mb), N50 values from 400 Kb to 18.9 Mb (average 3.8 Mb) complete BUSCO scores between 96.1% and 99%, and per base quality values (QV scores) between 37.2 and 52.9 (Table 1 and Supplementary Notes 2 – 4 ). CUSCO scores, i.e., percentage of contiguously assembled piRNA clusters 36 , range from 35.3% to 84.7% (average 64.1%; Table 1 ). The detectability of a cluster was inversely correlated with its size (Pearson´s correlation = −0.47; Supplementary Data 3b , Supplementary Fig.…”

“…We first masked simple repeats in both genomes using RepeatMasker (v.3.0) ( www.repeatmasker.org ) and then used MUMmer (v.3.0) 90 for genome alignment. The quality of the genomes in the context of TEs was evaluated using CUSCO (downloaded on May 6, 2020) (Cluster BUSCO; Wierzbicki et al 36 based on the flanking sequences for 85 out of the 142 annotated piRNA clusters of D. melanogaster 91 Supplementary Data 3b , Supplementary Note 5 ). QV scores were estimated according to Solares et al 18 using both SNPs and INDELs called from the mapping of Illumina short-reads over the de novo assembled genomes.…”

Rapid evolutionary diversification of theflamencolocus across simulans cladeDrosophilaspecies

“…The necessity of any single cluster for TE suppression in the germline piRNA pathway is unclear, but likely redundant [23]. However, flamenco is thought to be the master regulator of the somatic support cells of the ovary, preventing gypsy elements from hopping into germline cells [19,42,45,46,48,72]. It is not redundant to other clusters, and insertion of a single element into flamenco in D. melanogaster is sufficient to initiate silencing.…”

“…A summary of the assembly statistics is available in Supplementary Table 1. The quality of cluster assembly was evaluated using the coverage and soft clip quality as described in (20, 49) (Supplementary File 1).…”

“…The quality of cluster assembly was evaluated using the coverage and soft clip quality as described in (20,49) (Supplementary File 1).…”