The merits of the FLc-Capture method
The first advantage of the FLc-Capture method is that it saves both cost and time compared to commercially synthesized probe sets. Previous transcriptome-based capture studies normally used transcriptome data to design capture probes and ordered those probe sets from commercial companies (e.g., Bi et al., 2012; Bragg et al., 2016; Portik et al., 2016; Quek et al., 2020). The whole process to synthesize a custom probe kit typically takes several weeks and cost $2,400-$5,000, depending on the supplier (Peñalba et al., 2014). Although these commercial probes can be diluted for applying to more samples, the cost of using commercial probes would still be high when a research project has hundreds of samples or more, probably reaching several tens of thousands of dollars. In contrast, the primary initial investment for our method was the SMARTer PCR cDNA synthesis reagent (Clontech Inc.), which costs ~$80 per reaction. Including the extraction of RNA, the probe preparation can be done within three days. In our lab, one SMARTer PCR cDNA synthesis reaction can produce up to 100 µg of full-length cDNA probes when input RNA is 1 µg. Such amount of cDNA probes is enough to handle at least 2,000 samples.
FLc-Capture has the merit of transcriptome sequencing while largely avoiding its shortcomings. Compared to transcriptome sequencing, FLc-Capture can produce transcriptome-level data (thousands of ORF and UTR sequences) with only DNA samples. Researchers just need to collect one common species of their taxonomic group of interest for RNA extraction, prepare biotinylated full-length cDNA probes from RNA, and then use these probes to capture target regions from their DNA libraries. Except for the probe species used for RNA extraction, FLc-Capture has no strict requirements on the DNA quality of other samples, so highly degraded DNA extracted from old museum specimens can also be analyzed, which can greatly increase the sampling number of taxa in a phylogenomic study.
In addition, FLc-Capture uses cDNA sequencing to provide reference sequences. This feature enables researchers to efficiently capture thousands of coding and noncoding sequences without knowing any genome knowledge of the taxa been investigated, especially suitable for nonmodel organisms. Unlike low-coverage WGS sequencing, which is more suitable for extracting phylogenomic data from small genome species (Fig. 3), FLc-Capture can efficiently collect coding and noncoding phylogenomic data not only from small genome species but also large genome species. These two features make the FLc-Capture method highly versatile and applicable for any organism groups.