DOI: 10.5061/dryad.gmsbcc2qj
title: Data for: The role of repetitive DNA in re-patterning of major rDNA clusters in Lepidoptera

description: Genes for major ribosomal RNAs (rDNA) are present in multiple copies
 organized in tandem arrays. Number and position of rDNA loci can change
 dynamically and their re-patterning is presumably driven by repetitive
 sequences. We explored a peculiar rDNA organization in several
 representatives of Lepidoptera with either extremely large or numerous
 rDNA clusters. We combined molecular cytogenetics with analyses of second
 and third generation sequencing data to show that rDNA spreads as a
 transcription unit and reveal association between rDNA and various
 repeats. Furthermore, we performed comparative long read analyses between
 the species with derived rDNA distribution and moths with a single rDNA
 locus, which is considered ancestral. Our results suggest that satellite
 arrays, rather than mobile elements, facilitate homology-mediated spread
 of rDNA via either integration of extrachromosomal rDNA circles or ectopic
 recombination. The latter arguably better explains preferential spread of
 rDNA into terminal regions of lepidopteran chromosomes as efficiency of
 ectopic recombination depends on proximity of homologous sequences to
 telomeres.
description: Repeat Explorer analysis For analysis of
 repetitive DNA content, whole gDNA was sequenced on the Illumina platform
 generating either 150 bp pair-end reads from library with mean insert size
 450 bp (Novogene Co., Ltd., Beijing, China) or 250 bp PE reads with the
 mean insert size 700 bp in case of C. ohridella (Genomics Core Facility,
 EMBL Heidelberg, Germany). The raw reads were quality filtered and trimmed
 to uniform length of 120 bp (230 bp for C. ohridella) by Trimmomatic 3.2
 (Bolger et al., 2014). Random sample of two million (one million for C.
 ohridella) trimmed PE reads was analysed by RE pipeline (version
 cerit-v0.3.1-2706) implemented in Galaxy environment
 (https://repeatexplorer-elixir.cerit-sc.cz/galaxy/) with automatic
 annotation via blastn and blastx using the Metazoan 3 Repeat Explorer
 database. The resulting html files were searched for clusters annotated as
 major rDNA and their connection to other clusters. Long
 read sequencing and analysis High molecular weight DNA
 from H. humuli was enriched for fragments longer than 10 kbp by Short Read
 Eliminator (Circulomics Inc). The library was prepared by Ligation
 Sequencing Kit SQK-LSK110 (Oxford Nanopore Technologies, Oxford, UK)
 according to the manufacture’s protocol and therein recommended third
 party consumables. The library was snap-frozen and stored over night at
 -70°C and then sequenced using flowcell R10.3 and MinION Mk1B (Oxford
 Nanopore Technologies). Reads were basecalled by guppy 4.4.1. with high
 accuracy flip-flop algorithm. The data was filtered for reads 15kbp and
 longer with quality score over 10 using NanoFilt (De Coster et al.,
 2018). Quality and length filtered reads were searched
 for presence of major rDNA using blastn. Reads containing at least 1000 bp
 of H. humuli major rDNA unit were assembled by Flye 2.8 (Kolmogorov et
 al., 2019) using minimal overlap 8 kbp. The annotation of MEs was done by
 RepeatMasker 4.1.2-p1 (Smit et al., 2013) protein-based masking. Tandem
 repeats were identified based on self Dotplot implemented in Geneious
 11.1.5. Consensus sequences of all identified ME fragments together with
 major rDNA unit were mapped to individual rDNA bearing nanopore reads
 using minimap2 (Li, 2018) with appropriate pre-set. The presence and
 relative localization of individual elements was evaluated via R script (R
 version 4.0.3 in Rstudio version 1.4.1103) ,  only regions with mapping
 quality at least 20 were considered. Phymatopus
 californicus gDNA was sequenced on Oxford Nanopore platform in Novogene
 Co.,Ltd. PacBio HiFi reads of I. io  (project PRJEB42130) and A. urticae
 (project PRJEB42112) were obtain through the Darwin Tree of Life project
 (http://www.darwintreeoflife.org). PacBio CLR data were obtained from
 Sequence Read Archive (SRA) database (S. frugiperda SRR12642577; L. dispar
 SRR13505170-6, SRR13505182-3, and SRR13505187; P.xylostella SRR13530960).
 Further, the reads were processed same as in H. humuli except for the HiFi
 reads, which were not quality filtered. Similar
 approach to detect rDNA and associated repetitive DNA was used also in A.
 urticae chromosomal level genome assembly (Bishop et al., 2021) (ENA acc.
 No. PRJEB41896). Coverage analysis
 Coverage analysis was done by aligning genomic Illumina
 sequencing reads from H. humuli  I. io, and A. urticae to consensus
 sequences, which were generated by overlapping the contigs from RE in
 Geneious 11.1.5 or by Flye 2.8 assembler, using Bowtie2 aligner (Langmead
 et al., 2019; Langmead and Salzberg, 2012). Coverage values were obtained
 using samtools depth (v 1.10) (Li et al., 2009) and plotted using a script
 in R (R version 4.1.0 in Rstudio Workbench Version 1.4.1717-3). Mean
 coverage of defined annotation blocks as seen in Figure 3 was computed
 using R and is in Suppl. Tables 3.
description: Au_assembly_rDNA.fasta: A. urticae rDNA region assembly
 Au_rDNA_reads.fasta: A. urticae PacBio reads containing at least
 200 bp rDNA based on blast results
 Au_sat_only_reads.fasta: A. urticae PacBio reads containing at
 least 200 bp AuSat, however, no rDNA or othe IGS parts
 Hh_assembly_rDNA.fasta: H. humuli rDNA region assembly
 Hh_rDNA_reads.fasta: H. humuli Oxford Nanopore reads containing
 at least 200 bp rDNA based on blast results
 Ii_assembly_rDNA.fasta: I. io rDNA region assembly
 Ii_rDNA_reads.fasta: I. io PacBio reads containing at least 200
 bp rDNA based on blast results
 Ii_sat_only_reads.fasta: I. io PacBio reads containing at least
 200 bp IiSat, however, no rDNA or othe IGS parts 
 Ld_assembly_rDNA.fasta: L. dispar rDNA region assembly
 Ld_rDNA_reads.fasta: L. dispar PacBio reads containing at least
 200 bp rDNA based on blast results
 Pc_assembly_rDNA.fasta: P. californicus rDNA region
 assembly Pc_rDNA_reads.fasta: P. californicus Oxford
 nanopore reads containing at least 200 bp rDNA based on blast
 results Px_assembly_rDNA.fasta: P. xylostella rDNA
 region assembly Px_rDNA_reads.fasta: P. xylostella
 PacBio reads containing at least 200 bp rDNA based on blast
 results R_cov_analysis.html: R code used for illumina
 rDNA units coverage analysis R_rDNA_long_reads.html: R
 code used for long reads rDNA and associated repeats ananlysis
 Sf_assembly_rDNA.fasta: S. frugiperda rDNA region
 assembly Sf_rDNA_reads.fasta: S. frugiperda PacBio
 reads containing at least 200 bp rDNA based on blast results