DOI: 10.5061/dryad.rb1bt3j
title: Data from: Islands of retroelements are major components of Drosophila centromeres

description: Centromeres are essential chromosomal regions that mediate kinetochore
 assembly and spindle attachments during cell division. Despite their
 functional conservation, centromeres are amongst the most rapidly evolving
 genomic regions and can shape karyotype evolution and speciation across
 taxa. Although significant progress has been made in identifying
 centromere-associated proteins, the highly repetitive centromeres of
 metazoans have been refractory to DNA sequencing and assembly, leaving
 large gaps in our understanding of their functional organization and
 evolution. Here, we identify the sequence composition and organization of
 the centromeres of Drosophila melanogaster by combining long-read
 sequencing, chromatin immunoprecipitation for the centromeric histone
 CENP-A, and high-resolution chromatin fiber imaging. Contrary to previous
 models that heralded satellite repeats as the major functional components,
 we demonstrate that functional centromeres form on islands of complex DNA
 sequences enriched in retroelements that are flanked by large arrays of
 satellite repeats. Each centromere displays distinct size and arrangement
 of its DNA elements but is similar in composition overall. We discover
 that a specific retroelement, G2/Jockey-3, is the most highly enriched
 sequence in CENP-A chromatin and is the only element shared among all
 centromeres. G2/Jockey-3 is also associated with CENP-A in the sister
 species Drosophila simulans, revealing an unexpected conservation despite
 the reported turnover of centromeric satellite DNA. Our work reveals the
 DNA sequence identity of the active centromeres of a premier model
 organism and implicates retroelements as conserved features of centromeric
 DNA.
description: FileS1. Custom repeat
 libraryWe created a custom
 Drosophila-specific consensus repeat library modified from RepBase
 v20150807 to include all complex satellite DNAs from Drosophila
 melanogaster.File.S1.Chang_et_al.fastaFileS2. ChIPtigs from R1 libraryWe created de novo contigs from ChIPseq reads (ChIPtigs) with Spades v3.11.0 (-t 24 -careful –sc;) for the R1 library.File.S2.Chang_et_al.fastaFileS3. ChIPtigs from the R2 libraryWe subsampled reads from R2 ChIP-seq to 100x coverage using BBnorm (v37.54) with the parameters "threads=24 prefilter=t target=100", and created de novo contigs from the subsampled ChIPseq reads (ChIPtigs) with Spades v3.11.0 (-t 24 -careful –sc;).File.S3.Chang_et_al.fastaFileS4. ChIPtigs from the R3 libraryWe created de novo contigs from ChIPseq reads (ChIPtigs) with Spades v3.11.0 (-t 24 -careful –sc;) for the R3 library.File.S4.Chang_et_al.fastaFileS5. ChIPtigs from the R4 libraryWe created de novo contigs from ChIPseq reads (ChIPtigs) with Spades v3.11.0 (-t 24 -careful –sc;) for the R4 library.File.S5.Chang_et_al.fastaFileS6. ChIPtigs from the S2 libraryWe created de novo contigs from S2 ChIPseq reads (ChIPtigs) with Spades v3.11.0 (-t 24 -careful –sc;) from the S2 library.File.S6.Chang_et_al.fastaFileS7. Hybrid PacBio-Nanopore assemblyWe assembled nanopore (Solare et al. 2018) and PacBio reads (Kim et al. 2014) into a hybrid assembly using Canu v1.7 with default settings. The assembly size is 162,798,260 bp with N50=5,104,646 bp.File.S7.Chang_et_al.fastaFileS8. PacBio-only assembly with extra contigsWe use the PacBio-only assembly in Chang and Larracuente 2018 and added 19 sequences with CENP-A-enriched repeats. Of these 19 sequences, 6 were contigs from the hybrid PacBio-Nanopore assembly (File S7) and the rest were error-corrected PacBio reads.File.S8.Chang_et_al.fastaFileS9. Repeat annotation file for the finished PacBio-only assembly with extra contigsWe annotated the finished assembly using our custom repeat library (-lib library.fasta -s) and RepeatMasker 4.06.File.S9.Chang_et_al.gff.txtFileS10. Gene annotation file for the PacBio-only assembly with extra contigsWe transferred gene annotations from Flybase r6.20 to our genome using BLAT and CrossMap v0.2.5.File.S10.Chang_et_al.gffFile S11. The sequences of Stellaris probes for RspThe following Stellaris probes are tagged with Quasar 570 and used to detect Rsp sequences.File.S11.Chang_et_al.txtFile S12. The fasta alignment of genomic IGS sequences from D. melanogaster and outgroup speciesWe extracted all IGS elements from the genome using BLAST v2.7.1 with parameters “- task blastn -num_threads 24 -qcov_hsp_perc 90” and custom scripts. We then aligned and manually inspected IGS sequences using Geneious v8.1.6.File.S12.Chang_et_al.fastaFile S13. A fasta alignment of G2/Jockey-3 sequences from different contigs and outgroup speciesWe extracted the G2/Jockey-3 sequences based on Repeatmasker annotations and custom scripts. We then aligned and manually inspected G2/Jockey-3 sequences using Geneious v8.1.6.File.S13.Chang_et_al.fastaFile S14. The newick consensus tree of IGS sequences inferred using RAxMLWe constructed maximum likelihood phylogenetic trees for IGS using RAxML v.8.2.11 with parameters “-m GTRGAMMA -T24 -d -p 12345 -# autoMRE -k -x 12345 -f a”.File.S14.Chang_et_al.nwkFile S15. The newick consensus tree of G2/Jockey-3 sequences inferred using RAxMLWe constructed maximum likelihood phylogenetic trees for G2/Jockey-3 using RAxML v.8.2.11 with parameters “-m GTRGAMMA -T24 -d -p 12345 -# autoMRE -k -x 12345 -f a”.File.S15.Chang_et_al.nwkFile S16. Oligopaint coordinates and sequencesOligopaints sequences and information for centromeres X, 3, 4, and Y. The columns indicate the centromere contig ID, start and end coordinates of sequence, followed by the oligo sequence, and the melting temperature (all.oligos.cen.islands). Included are also the same Oligopaint sequences with 5’ and 3’ extensions containing the universal primer followed by library-specific barcodes (oligos.with.adaptors).File.S16.Chang_et_al.xlsx