DOI: 10.5061/dryad.4j0zpc8d3
title: Genome sequence and silkomics of the spindle ermine moth, Yponomeuta cagnagella, representing the early diverging lineage of the ditrysian Lepidoptera

description: Many lepidopteran caterpillars produce silk, cocoons, feeding tubes, or
 nests for protection from predators and parasites. Yet, the number of
 lepidopteran species whose silk composition has been studied in detail is
 very small, because the genes encoding the major structural silk proteins
 tend to be large and repetitive, making their assembly and sequence
 analysis difficult. Here we have analyzed the silk of Yponomeuta
 cagnagella, which represents one of the early diverging lineages of the
 ditrysian Lepidoptera thus improving the coverage of the order. To obtain
 a comprehensive list of the Y. cagnagella silk genes, we sequenced,
 assembled, and annotated the draft genome using Oxford Nanopore and
 Illumina technologies. The 626 Mb assembly with N50 of 96.5 kb contained
 96.9% insect orthologs recovered by BUSCO and 30,003 predicted gene
 models. We then used a silk-gland transcriptome and a silk proteome to
 identify major silk components and verified the tissue specificity of the
 expression of individual genes. 
description: To assemble the genome of
 <em>Y. cagnagella</em>, Oxford Nanopore reads were sequenced
 on the Nanopore PromethION platform. In addition, an Illumina library with
 a 700 bp insert size was prepared and sequenced on the Illumina HiSeq 2500
 with 250 bp paired-end reads. The raw reads were deposited in NCBI under
 SRA accession numbers SRR15714088 and SRR15714089.
 For genome annotation, RNA from heads, thoraces,
 and gonads of three male and female imagoes was extracted with
 TRI-Reagent. Biological replicas were pooled prior to isolation, resulting
 in three tissue-specific samples per sex. First, adaptor sequences and low quality
 bases were filtered out of the Illumina data using Trimmomatic. Similarly,
 Nanopore reads shorter than 500 bp and with a quality score lower than 7
 were removed from the dataset with NanoFilt. Next, the FM-index Long Read
 Corrector (FMLRC) was used with default settings to correct the long reads
 using the filtered Illumina sequences. As recommended, ropebwt2 and
 fmlrc-convert were used to construct the multi-string BWT data structure
 required by the FMLRC pipeline. The preprocessed long reads were then
 assembled with Flye. To
 eliminate the haplotypic duplications from the primary assembly,
 purge_dups pipeline was applied, followed by polishing using POLCA. Repeat
 composition and average GC content were analysed with RepeatModeler and
 RepeatMasker software packages. To achieve more accurate masking, major
 satellites were identified with TAREAN from Illumina data subsampled to
 0.25× genome coverage. A
 custom repeat library built from the genome sequence with RepeatModeler
 with added satellite dimers was used in RepeatMasker pipeline to survey
 the landscape of repetitive elements and generate a masked version of the
 <em>Y. cagnagella</em> assembly. To
 annotate the assembly, all RNA-seq data were concatenated into a single
 dataset, including the silk gland RNA-seq (see below) and the quality of
 the sequencing was verified using FastQC. The resulting data were aligned
 to the masked genome assembly using STAR. The genome index was generated
 with the following parameter scaled down to the size of <em>Y.
 cagnagella</em> genome: “--genomeSAindexNbases 13”. Genes were predicted with BRAKER and
 annotated using BLASTp with NCBI RefSeq invertebrate protein database, all
 implemented in the GenSAS platform. For analysis of silk genes, total RNA from
 the last larval instar silk glands was isolated using TRIzol reagent,
 followed by isolation of mRNA using Dynabeads Oligo (dT)25 mRNA
 Purification Kit, and cDNA was prepared using the NEXTflex Rapid RNA-Seq
 Kit. The cDNA library was sequenced on Illumina platform 2×150 bp
 (paired-end reads) with MiSeq. 150 bp paired-end Illumina reads were
 visually inspected for quality using FastQC. Adaptor sequences removal and
 trimming were performed using BBDUK. A further rRNA contamination step was
 conducted using BBDUK with the associated ribokmers.fa file to eliminate
 rRNA contamination from the mRNA enrichment step of library preparation.
 Cleaned reads were assembled into a transcriptome using the multi k-mer
 rnaSPAdes assembler. K-mer sizes of 25, 35, 45, 55, 65, and 75 were chosen
 for de novo assembly to increase the likelihood of maximum transcript
 recovery.