2.2 Genomic and transcriptomic sequencing
Genomic DNA was isolated from aphid samples using a DNeasy Blood & Tissue Extraction Kit (Qiagen Inc., Valencia, CA, USA) by following the manufacturer’s instructions. After the measurement of quality and quantity, the DNA samples were used for making a paired-end sequencing library (150 bp in length). The library was sequenced using the Illumina NovaSeq6000 platform for genome size assessment. In addition, a 20 kb library was constructed and sequenced using the PacBio RSII platform at Annoroad Gene Technology Co., Ltd. (Beijing, China). Hi-C libraries were also constructed from aphid samples according to the Proximo Hi-C procedure and its quality and concentration were determined via an Agilent 2100 Bioanalyzer and qPCR. Briefly, aphid samples were mixed with 1% formaldehyde for 10 min at room temperature and the nuclei were extracted and permeabilized. After the quality of the library was ascertained, different libraries were pooled to achieve required concentrations for Illumina sequencing (Rao et al., 2014).
Transcriptomes were generated from RNA samples extracted from the fundatrix, the fundatrigeniae, autumn migrants, nymphs, spring migrants (sexuparae), male and female sexuales, respectively, following the standard protocols provided by the manufacturer. RNA quantity, purity and integrity were determined by a NanoPhotometer and the Agilent 2100 Bioanalyzer. cDNA libraries were built following the chain specific method. The libraries were initially quantified with qubit 2.0 fluorometer and diluted to 1.5 ng/ul. Different libraries were pooled according to the requirements of effective concentration and target data volume for Illumina sequencing. Low-quality bases in the RNA-Seq raw reads were first filtered using Trimmomatic (version 0.36) (Bolger, Lohse, & Usadel, 2014). Then, the clean reads were mapped to the genome assembly using Hisat2 (version2.1.0.5) (Kim et al., 2015) to obtain putative transcripts. The transcripts gene expression was analyzed by using cufflinks (version2.2.1) (Ghosh, & Chan, 2016).