Nextera mate pair library

For whole genome sequencing of AP3_16^T strain, DNA was isolated following a previously described protocol (Furmanczyk et al., 2017a (link)). The isolated genomic DNA was used to prepare two types of libraries: (1) paired-end library with average insert size 500 bp (using KAPA HTP Library Preparation Kit for Illumina platforms according to manufacturer’s protocol) (Kapa Biosystems, United States), (2) Nextera^® Mate Pair library with average insert size 8 kbp (using Illumina protocol) (Illumina, United States). The libraries were verified using a 2100 Bioanalyzer (Agilent, United States) High-Sensitivity DNA Assay and KAPA Library Quantification Kit for the Illumina (Kapa Biosystems, United States). Sequencing was performed using an Illumina MiSeq (MiSeq Reagent Kit v3, 600 cycles) (Illumina, United States) with read length of 300 bp.

To provide a reference assembly for mapping reads from the ddRAD data, we generated libraries that were optimized to be assembled using the ALLPATHS‐LG assembler (Gnerre et al., 2011). We used flies collected from the rearing colony maintained at the Petapa San Miguel Mexfly Mass Rearing Facility in Guatemala, collected from the Family 10 black pupae genetic sexing strain, and extracted DNA as for the ddRAD specimens. We constructed a 180‐bp insert Illumina TruSeq fragment library from 500 ng DNA from a single female individual, and an Illumina Nextera mate‐pair library targeting a 3‐kb insert size from a pool of sibling male individuals (eight individuals in total), to have sufficient DNA for mate‐pair library construction. The fragment and mate‐pair libraries were each sequenced on a lane of Illumina HiSeq4000 with 2 × 100 bp paired‐end sequencing and 2 × 50 bp paired‐end sequencing, respectively. We constructed a scaffold assembly from raw reads of both libraries using ALLPATHS‐LG (Gnerre et al., 2011). We performed k‐mer‐based error correction (using the ALLPATHS‐LG pipeline) to the fragment library and then ran the pipeline with default settings except for the addition of the “HAPLOIDIFY = TRUE” parameter.

DNA isolation followed a modified version of Georgi et al.62 (link). An Illumina 300 bp insert TruSeq V2 library was prepared and sequenced by the Center for Genome Research at Oregon State University (http://cgrb.oregonstate.edu/) and sequenced on a HiSeq 2000 (2 × 100 bp sequencing on ⅓ of a lane). High quality reads were filtered and trimmed using Trimmomatic V0.3456 (link) using: ILLUMINACLIP:2:30:10 and LEADING:25 TRAILING:25 SLIDINGWINDOW:5:25 MINLEN:65.
A linear plastome assembly including just one IR was generated using the Geneious v6.1.6 (Biomatters, Auckland NZ) as the de novo assembler and 3 M read-pairs of data (ca. 100X coverage). Subsequently, all 30 M read-pairs were mapped to the genome to correct errors and confirm the full genomic sequence. Independently assembled de novo IR boundary regions were reciprocally mapped to draft IR boundaries to confirm draft boundaries. These were confirmed using the BLAST-on-BLAST method discussed below (see “dispersed repeats”). The structural organization of the plastome assembly has since been confirmed by the addition of a 4 kb insert Illumina Nextera mate pair library developed and sequenced (2 × 100 bp reads) by the University of Maryland’s Genomic Resource Center. The annotated plastome sequence has been deposited in GenBank (KT428297) and the Illumina reads are in SRA305491.