Constructing reference genome sequence files for particularly uncommon species can be challenging due to the limited availability of reference genomes and genome annotation data. However, there are several methods that can assist in this process:


Based on known related species: Utilize genome sequence alignment and assembly techniques to construct the reference genome sequence of the target species, leveraging the known genome sequences of closely related species. This method requires sufficiently similar genome sequences and adequate alignment depth.


RNA-Seq data assembly: If RNA-Seq data of the target species is accessible, assemble the transcriptome sequence using RNA-Seq reads. Then, employ tools like BLAST and STAR to assemble and annotate the genome based on the transcriptome sequence. This approach is suitable when a complete genome sequence is not necessary.


Subgenomic annotation: In the absence of genomic data, consider subgenomic annotation methods. This involves mapping genome annotation information from known species to the target species using tools such as BLAST and HMMER. Consequently, the genome composition and structure of the target species can be inferred.


Third-generation sequencing-assisted assembly: Consider employing faster and more accurate third-generation sequencing technologies like Oxford Nanopore and PacBio SMRT. These technologies generate long-read sequencing data, which aids in improved genome assembly and annotation.