Clc genomic workbench v 8

Total RNA was isolated from stomach tissue using the TRIzolTM reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The concentration and integrity numbers of purified RNA samples were assessed by Qubit 2.0 fluorometer (Thermo Fisher Scientific) and 2200 TapeStatio (Agilent Technologies, Santa Clara, CA, USA), respectively. The targeted sequencing and transcriptome sequencing were performed following the protocol reported in previous studies [45 (link)]. Briefly, Sequencing-ready libraries of amplified targeted genes were prepared using TruSeq targeted RNA Wnt pathway panel kit (Illumina, San Diego, CA, USA) by following the manufacturer’s manuals [46 ]. Sequencing was carried in a MiSeq sequencer (Illumina), and a DESeq package was used to remap the sequencing reads to the human genome (hg19). The expression level of each gene was represented by gene read number/total specimen read number. The libraries for transcriptome sequencing analysis were prepared using Agilent SureSelect strand-specific mRNA library preparation kit following the manufacturer’s directions and were sequenced in 100-bp paired-end reads on Illumina MiSeq. Mapping, annotation and calculation of gene expression level (fragments per kilobase of transcript per million mapped) were performed using CLC Genomic Workbench v. 8.5 (Qiagen, Hilden, Germany).

Analysis of the 16S rRNA gene amplicons was carried out essentially as previously described (31 (link)). In brief, sequences were demultiplexed and trimmed to remove barcodes and primers, and sequences below 125 bp or above 180 bp were discarded using the CLC Genomic Workbench (v8.5) software (CLCbio, Qiagen, Aarhus, Denmark). Using the DADA2 pipeline v1.12.44 in the R software (https://www.R-project.org/), reads were quality filtered (maxEE = 1, maxN = 0, truncQ = 2)and denoised (homopolymer gap penalty = −1, band size = 32), and chimeric sequences were removed. Amplicon sequence variants (ASVs) were assigned taxonomy using the RDP 16S rRNA database (rdp_train_set_18.fa.gz). Using QIIME2 (2019.1), ASVs with a frequency of <100 reads across all samples or assigned to Cyanobacteria/chloroplast were discarded. The core diversity metrics function was used with a rarefaction depth of 8,300 reads per sample to create Bray-Curtis distance matrices and alpha diversity measures (Shannon index). Using the rarefy table function, the ASV table was rarefied to 8,300 reads per sample and collapsed to genus levels using the taxa collapse function. Relative abundances at genus level were calculated by total sum scaling normalization.

For the assembly of the Helicoverpa mitogenomes (Table 1), we used two separate methods involving different assembly programs of either the genomic software Geneious R8 version 8.1.9 (Biomatters Pty Ltd., Auckland, NZ) or the CLC Genomic Workbench v8.5 (Qiagen). The mitogenome of H. assulta from Thailand was de novo assembled using CLC Genome Workbench, and the remainder of the mitogenomes was assembled using mitogenomes of H. punctigera (KF977797), H. armigera (GU188273), and H. zea (KJ930516) as reference sequences. After the initial sequence assembly based on the appropriate reference mitogenome, we reassembled these mitogenomes against their first version mitogenome templates. With each subsequent reassembly, we fine‐tuned and removed all ambiguity by manually checking for potential misassembled regions. This procedure was repeated between three to eight times until a complete draft mitogenome was obtained. Draft mitogenomes from this study are available in GenBank (MG437189‐MG437202, KT626655; Table 1).