Hiseq 2500 lane

As with the other RNA-seq libraries, these libraries were prepared using the TruSeq RNA Sample Preparation Kit (Illumina, catalog no. FC-122-1001). For the polyA sample, 1 μg of IVT RNA was treated with polyA selection reagents included with the TruSeq RNA Sample Preparation Kit according to the manufacturer’s protocol. The remainder of the library preparation was carried out using the same conditions as for the other IVT RNA samples. For the no selection sample, 100 ng of IVT RNA at a concentration of 100 ng/μL was diluted with 17 μL of Elute, Prime, Fragment mix (provided with the TruSeq RNA Sample Preparation Kit). Again, the remainder of the library preparation was carried out as with the other samples. These samples were sequenced in a single Illumina HiSeq 2500 lane (paired 100 bp reads).

Due to the discovery of limited mitochondrial variation, we conducted whole-genomic sequencing on Dactylopius coccus to better understand domesticated cochineal phylogenies. Three bulk extracts representing cochineal raised by Oaxacan small-scale farmers or sold by Mexican and Peruvian commercial vendors were subjected to Pool-Seq (Schlötterer et al. 2014 (link); Fig.1). Bulk DNA was extracted from 50 individuals each (Schlötterer et al. 2014 (link); Appendix). Sequencing libraries were prepared from the bulk extracts using the PrepX Illumina Kit (IntegenX, Pleasanton, California, USA) and NEXTflex™ DNA Barcodes (Bioo Scientific, Austin, Texas, USA) on the Apollo 324 robotic platform (IntegenX). Paired-end 150-bp sequences were generated on one-quarter of an Illumina HiSeq 2500 lane. A total of 5.2–5.5 million paired sequences were obtained per library.

Targeted enrichment of HPV DNA was performed using hybridization capture probes homologous to the full‐length genomes of 15 different HPV types (6, 11, 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, 68, 69) that were custom‐designed using the Roche Nimblegen SeqCap EZ System (Roche, Basel, Switzerland). Biotinylated oligonucleotide probes specific for each HPV type were designed at ~150 bp intervals encompassing the positive strands of the complete ~8 kb double strand viral genomes to achieve 100% coverage. For library preparation, tumor genomic DNA was mechanically fragmented to 200 bp (Covaris, Woburn, MA), and Illumina adaptors were ligated at each end. Libraries were then hybridized to the custom HPV capture probes for 72 h using the Roche target enrichment protocol following manufacture instructions and sequenced on one Illumina HiSeq 2500 lane (Illumina, San Diego, CA) using the paired end 150 bp sequencing mode. After sequencing, we aligned adaptor‐cleaned, QC‐passed, de‐duplicated, paired end reads to a custom human (GRCh37/hg19) plus HPV reference genome containing 143 alpha genus HPV types from the Papillomavirus Episteme48 using Burrows‐Wheeler Aligner (BWA).49 Junction fragments were computationally identified using a combination of programs; Delly50 and SplazerS,51 both of which specify a combined read pair discordancy and split read analysis.

DNA was extracted from a week-old freeze dried seedlings representing one of the five tagged plants of 1425 accessions following acetyl trimethylammonium bromide (CTAB) protocol modified for 96-well plates (Mace et al., 2003 (link)). A total of fifteen 96-plex GBS libraries were constructed and genotyped at the University of Wisconsin, Biotechnology center. The genotyping by sequencing (GBS) procedure (Elshire et al., 2011 (link)) was implemented using the ApeKI restriction enzyme (recognition site, G| CWCG). The GBS library was sequenced on Illumina HiSeq 2500 lane following the manufacturer’s protocol. The SNP datasets generated for this study can be found in Purdue university repository².