2.2 Genomic and transcriptomic sequencing
Genomic DNA (gDNA) was isolated from 200 individual female and male
using the DNeasy Blood & Tissue Extraction Kit (Qiagen Inc., Valencia,
CA, USA), following the manufacturer’s instructions. After quality and
quantity measurements, the gDNA was used to construct a 150-bp
paired-end sequencing library for Illumina platform. A 20 kb long-read
sequencing library was constructed by gDNA isolated from 200
fundatrigeniae for PacBio Sequel II platform.
For Hi-C analysis, 200
fundatrigeniae were soaked in 1%
formaldehyde for 10 min at room temperature and in a 2.5 M-glycine
solution to terminate the isolation and cross linking of aphid cells.
The Hi-C assays and the sequencing procedures were performed via a
commercial contract with Annoroad Gene Technology Co., Ltd. (Beijing,
China) (Rao et al., 2014).
Transcriptomes were generated from
RNA samples extracted from different stages including fundatrix,
fundatrigeniae, autumn migrants, nymphs, spring migrants (sexuparae),
male and female sexuales, separately. RNA quantity, purity and integrity
were determined on a NanoPhotometer and an Agilent 2100 Bioanalyzer.
cDNA libraries were subsequently constructed following the chain
specific method. The libraries were initially quantified by the qubit
2.0 fluorometer and diluted to 1.5 ng/ul. Later, 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 filtered using Trimmomatic (version 0.36)
(Bolger, Lohse, & Usadel, 2014). Clean reads were mapped to the genome
assembly using Hisat2 (version 2.1.0.5) (Kim et al., 2015), so as to
obtain the putative transcripts. Transcript levels were analyzed using
cufflinks (version 2.2.1) (Ghosh, & Chan, 2016).