Simultaneously Collecting Coding and Noncoding Phylogenomic Data Using
Homemade Full-length cDNA Probes, Tested by Resolving the High-level
Relationships of Colubridae
Abstract
Resolving intractable phylogenetic relationships often requires
simultaneously analyzing a large number of coding and noncoding
orthologous loci. To gather both coding and noncoding data, traditional
sequence capture methods require custom-designed commercial probes.
Here, we develop a cost-effective sequence capture method based on
homemade probes, to capture thousands of coding and noncoding
orthologous loci simultaneously, suitable for all organisms. This
approach, called “FLc-Capture”, synthesizes biotinylated full-length
cDNAs from mRNA as capture probes, eliminates the need for costly
commercial probe design and synthesis. To demonstrate the utility of
FLc-Capture, we prepared full-length cDNA probes from mRNA extracted
from a common colubrid snake. We performed capture experiments with
these homemade cDNA probes and successfully obtained thousands of coding
and noncoding genomic loci from 24 Colubridae species and 12 distantly
related snake species of other families. The average capture specificity
of FLc-Capture across all test snake species is 35%, similar to the
previously published EecSeq method. We constructed two phylogenomic data
sets, one including 1,075 coding loci (~817,000 bp) and
another including 1,948 noncoding loci (~1,114,000 bp),
to study the phylogeny of Colubridae. Both data sets yielded highly
similar and well-resolved trees, with 85% of nodes having
> 95% bootstrap support. Our experimental tests indicated
that FLc-Capture is a flexible, fast, and cost-effective sequence
capture approach for simultaneously gathering coding and noncoding
phylogenomic data sets to study intractable phylogenetic questions. We
anticipate that this method can provide a new data collection tool for
the evolutionary biologists working in the era of high-throughput
sequencing.