Genome Assembly Using Illumina Sequencing

The Illumina platform (Miseq and Hiseq 2500) was used for sequencing.¹³ (link) Libraries were prepared according to slight modifications of protocols provided by the manufacturer. Fragmented genomic DNA was further purified using Blue Pippin (Sage Science). A paired-end library consisting of clones ∼720 bp was prepared for the Miseq using a TruSeq DNA PCR-Free LT Sample Prep Kit (Illumina), and 3-kb and 8-kb mate-pair libraries were prepared for the Hiseq 2500 using a Nextera Mate Pair Sample Prep Kit (Illumina), respectively (Supplementary Table S1). Longer reads were obtained by using more reagent kits for the Hiseq. K-mer counting and estimation of genome size were performed using JELLYFISH 2.2.0 software.¹⁴ (link)^,15 (link)
Adapter sequences were trimmed from all reads using Trimmomatic-0.30.¹⁶ (link) Paired-end reads of high quality (quality value ≥ 20) were assembled de novo using Newbler 2.9 (GS Assembler) to create contigs. Then subsequent scaffolding of the Newbler output was performed using SSPACE 3.0,¹⁷ (link) based on Illumina mate-pair information. Gaps inside scaffolds were closed using GapCloser 1.12.¹⁸ (link) Diploid sequences of gap-closed scaffolds were merged with Haplomerger-2-20151124.¹⁹ (link) CEGMA 2.5 software²⁰ (link) was used to evaluate genome assembly. The mitochondrial genome was generated with the IDBA_UD 1.1.1 assembler.²¹ (link)