Collected from Rothamsted Farm and Cross Farm in Harpenden during Oilseed Rape harvest in July 2020. Cross Farm GPS: 51.803468, -0.331117.
Next Generation Sequencing
i) Illumina Hi-C sequencing 150 bp paired end data:
290,805,515 reads and 64x coverage.
ii) Illumina RNA-seq sequencing 150 bp paired end data, totalling 193,174,246 reads.
iii) PacBio HiFi data, of mean read length 11,109, total reads 1,215,508, read length N50 12,165, and total bases 13,503,462,642. DNA was extracted using MagAttract at the University of Delaware (600ng gDNA).
Non-sexed single individual DNA used for PacBio HiFi and multi-individual for Hi-C Illumina sequencing (University of Delaware USA, Arima Genomics USA respectively). Hifiasm was used to assemble the PacBio HiFi, with Juicer then 3d-dna using Hi-C data for chromosome level assembly. Haplotigs were removed (purge_haplotigs). Unmapped reads were mapped back to the original assembly to check for missing sequence and incorporated into the final assembly. Manual curation was done to bring the genome together and check for miss-assemblies.
PGI RNA-seq was assembled into a transcriptome (BUSCO: C:98.0%[S:95.0%,D:3.0%],F:0.7%,M:1.3%) and used in the Maker2 annotation pipeline with trained Augustus and Genemark gene predictors. PASA was used to update the gene models to add UTR, correct existing models and add isoforms. Non-coding RNA was annotated using Infernal v1.1.4.
A Pfam genomic track was created by converting to six reading frames and utilizing hmmer to identify loci of interest i.e. P450 pfam domains on the genome. Using this information, loci of interest including UDP, P450, ABC and IRAC gene models were found and curated using mapped RNA-seq and a Maker gene annotation.
Two Endosymbionts were assembled which included Rickettsia (4,063,491 bp) and Wolbachia (1,506,389 bp).