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.