DNA/RNA isolation and sequencing

For whole genome analyses, nematodes were propagated on NGM plates implemented with E. coli Op50 strain. After 2 weeks of incubation at 20 °C, nematodes were collected from the plate, washed five times with M9 buffer and the genomic DNA was extracted using Genomic-tip (Qiagen) following the manufacturer’s protocol. Paired-end and Mate-pair sequencing libraries were prepared using the Nextera DNA Sample Prep kit (Illumina) and TruSeq DNA Library Preparation kit, respectively, according to the manufacturer’s instructions and sequenced using Illumina MiSeq sequencer with the v3 kit (301 cycles × 2 or 76 cycles × 2) (Illumina) (Supplementary Table ^S2).

Two μg of genomic DNA was used to prepare Nanopore sequencing library using the Ligation Sequencing Kit SQK-LSK109 (Oxford Nanopore Technologies) according to the manufacturer’s protocol. The library was sequenced with a single 24 h run with FLO-MIN106 R9 MinION flowcell (Oxford Nanopore Technologies). Base calling for R9 runs was performed with Guppy v.3.1.5 using the ‘dna_r9.4.1_450bps_fast’ model and obtained 771,594 reads (~ 3 Gb) (Supplementary Table ^S2).

For mRNA-seq analysis, RNA was extracted from fresh mixed-stage nematodes using TRI reagent according to the manufacturer’s instructions. Total RNA samples were qualified using Bioanalyzer 2100 (Agilent Technology, Inc.) and only samples with an RNA integrity value (RIN) greater than 8.0 were used for library constructions. One hundred ng of total RNA was used to produce an Illumina sequencing library using the TruSeq RNA-seq Sample Prep kit according to the manufacturer's recommended protocols (Illumina). The RNA libraries were sequenced using Illumina MiSeq sequencer with the v3 kit (301 cycles × 2) (Illumina) (Supplementary Table ^S2).