4th or 5th instar larvae were collected on 25.03.2020; USA; 700 Chesterfield Pwky W Chesterfield, MO 63017 (Bayer).
Next Generation Sequencing
i) Illumina Hi-C sequencing 150 bp paired end data:
770,011,116 reads and 115,501,667, 400x coverage.
ii) PacBio HiFi data, of mean read length 9,416, total reads 508,419, read length N50 10,270, and total bases 4,787,652,669.
iii) PacBio CLR data, of mean read length 8,358, total reads 10,515,233, read length N50 11,110, and total bases 87,889,471,906. DNA was extracted for both CLR and HiFi using a single individual with a ZYMO HMW kit (6000ng gDNA).
iiii) PacBio isoseq data, x2 smrt cells using Sequel II.
Non-sexed single individual’s DNA used for PacBio CLR and HiFi (University of Georgia, USA) and multi-individual for Hi-C Illumina sequencing (Arima Genomics USA). Flye was used to assemble the PacBio CLR and Hifiasm the HiFi, The HiFi assembly was used with Juicer then 3d-dna using Hi-C data for chromosome level assembly. Haplotigs were removed (purge_haplotigs). Manual curation was done to bring the genome together and check for miss-assemblies. Unmapped reads were mapped back to the original assembly to check for missing sequence and incorporated into the final assembly. Error correction was done with Hi-C data using freebayes.
Public RNA-seq PRJNA564321 (mid-gut), PRJNA527774 (larval paratization), transcriptome was assembled (BUSCO: C:94.7%[S:22.9%,D:71.8%],F:1.1%,M:4.2%) and used with the PGI ISO-seq transcriptome (1 smrt cell) (BUSCO C:69.1%[S:36.7%,D:32.4%],F:4.6%,M:26.3%) 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.