Application suggestions of the FLt-Capture method
In
our FLc-Capture experiment, we did not perform cDNA normalization to
decrease the abundance of highly expressed transcripts, so the cDNA
probe pools skew towards highly expressed genes. However, our
FLc-Capture result showed that the capture depth of the obtained ORF and
UTR sequences is not all related to the abundance of their cDNA probe
(Fig. 5b); using unnormalized cDNA probes is still able to capture
thousands of coding and noncoding loci. To obtain more uniform capture
coverage across high and low-expressed transcripts, Puritz and Lotterhos
(2017) used duplex-specific nuclease (DSN) treatment to prepare
normalized cDNA probes for exome capture. They found that RNA sequencing
coverage and exome sequencing coverage was still highly correlated
(capture coverage will be higher for highly expressed genes) even using
normalized cDNA probes. Therefore, it seems that the cDNA normalization
is not an indispensable step. For projects focused on obtaining more
phylogenetically informative loci for phylogenomic analysis, increasing
the diversity of cDNA probes may be more effective than cDNA
normalization. In such scenarios, we suggest to pool mRNAs extracted
from multiple tissue types to create a high-diversity probe pool rather
than using only liver mRNA as in our demonstration case.
Despite the demonstrated
effectiveness of FLc-Capture from our snake case, one note should be
considered before employing the method. Because the efficiency of
sequence capture decreases with increased genetic distance between the
probes and the targets, FLc-Capture might be less effective in large and
highly divergent organism groups such as insects and other arthropods.
In our demonstrating snake case, the maximal sequence difference between
our probe species and outgroup species is about 15%, and we finally
recovered 65% of the target ORF loci and 35% of the target UTR loci
from these outgroup species, respectively. This threshold value (15%
genetic difference) can be used as a starting point for other
researchers to determine the phylogenetic depth of their FLc-Capture
experiments. It has been shown that
using DNA mixtures pooled from different representative species to
prepare homemade probes is an effective strategy for sequence capture
across large phylogenetic scales (Zhang et al.,
2019). Therefore, if an
investigator wants to apply the FLc-Capture method to a highly divergent
organism group, it is possible to use several probe species that cover
the entire phylogenetic span, mix their mRNAs to prepare a full-length
cDNA probes, which can reduce the sequence divergence between probe and
target. Of course, in such circumstances, the reference ORF and UTR sets
should also be separated by different probe species, and the
bioinformatics pipeline should be adjusted accordingly. This mixing
strategy may allow for applying FLc-Capture across large phylogenetic
scales but needs to be tested in the future.