Hybrid Genome Assembly Using Illumina and PacBio

Sequencing was performed using the Illumina MiSeq platform and MiSeq Reagent Kit v2 (300 cycles), with MiSeq Reagent Kit v3 (600 cycles) being used for a more detailed analysis. Libraries for paired-end and mate-pair sequencing were prepared using a TruSeq DNA PCR-Free Sample Prep Kit and Nextera Mate Pair Sample Prep Kit (Illumina, San Diego, CA, USA), respectively. The generated reads were quality-filtered using Prinseq (36 (link)), and then assembled into contigs and scaffolds using SPAdes 3.9.0 (5 (link)). Gaps within and between scaffolds were closed by PCR amplification and sequenced using the ABI3730 Genetic Analyzer. In addition, sequence reads of 4–5 kb were generated on the PacBio RSII platform using a BluePippin DNA Size-Selection system (Sage Science, Beverly, MA, USA) and P6-C4 chemistry (Pacific Biosciences, Menlo Park, CA, USA). PacBio reads were used to verify the assembly and circularity of the scaffolds by mapping the reads using the BLASTn program of BLAST+ suite (8 (link)). MiSeq-read coverage was calculated by counting k-mers covering each scaffold in the assembly using SPAdes 3.9.0.