Reconstructing Chloroplast Genomes from Illumina Sequencing

We constructed genomic libraries using the TruSeq DNA Nano kit with a DNA insert size of 350 bp. Sequencing was conducted on the Illumina X Ten platform, which generated at least 21 Gb raw data from each species. Sequence data were quality trimmed using SOAPnuke (with the options: -n 0.1 -| 20 –q 0.1 -5 1) (Chen et al., 2018 (link)). Sequences were assembled into contigs according to the protocol described by Hahn, Bachmann & Chevreux (2013) (link) using MIRA sequence assembler software (default settings) with the reference genome I. trifida (accession number: KF242476.1). The reads were blasted into contigs with perl scripts and contigs were extended to obtain the complete sequence. Finally, we blasted the reads to the complete sequence, detected the coverage of reads and manually corrected the sequence. The services of library construction, sequencing, and assembly were provided by Macrogen (http://www.macrogencn.com/sy, Shenzhen, China).
The eight chloroplast genome sequences were initially annotated using the online CpGAVAS (Liu et al., 2012 (link)) software with default settings, and then manually corrected using Genious 11.0.5. The circular chloroplast genome maps were constructed using the OrganellarGenome DRAW tool (Lohse, Drechsel & Bock, 2007 (link)).