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).