Materials and methods
Experimental overview
FLc-Capture sequencing is designed with two specific goals: (a) to eliminate the need for expensive capture probe synthesis and (b) to obtain genome-scale data of both coding and noncoding regions simultaneously. To this end, researchers should choose one common species readily available from their taxonomic group of interest to extract high-quality RNA and synthesize full-length cDNAs. These full-length cDNAs are then amplified by biotinylated primers to generate the homemade capture probes for subsequent sequence capture experiments. The steps for probe preparation and sequence capture are visualized in Figure 1.
Taxon sampling, DNA extraction, and library preparation
The family Colubridae is a widespread snake group with relatively large genomes (~2 G). It is a rapid radiation lineage and the subfamily relationships within this family has historically proven difficult to resolve. Therefore, the family Colubridae is a good case study for demonstrating the utility of the FLc-Capture method for generating genome-scale coding and noncoding data sets to resolve difficult phylogenetic questions. Based on the latest phylogenies of Colubridae (Li et al., 2020), we sampled 24 colubrid species representing 23 genera, seven subfamilies (Dipsadinae, Pseudoxenodontinae, Natricinae, Sibynophiinae, Calamariinae, Ahaetuliinae, Colubrinae) and 12 distantly related outgroup snake species from five families (Xenodermatidae, Pareatidae, Viperidae, Elapidae, Homalopsidae). The detailed information of these samples, such as taxonomy, collection locality, and voucher, is given in Table 1.
Total genomic DNA was extracted from ethanol-preserved liver or muscle tissue of each sample using a TIANamp Genomic DNA Kit (Tiangen, Beijing). All DNA extracts were measured using an ND-2000 spectrophotometer and diluted to a concentration of 50 ng/μl with 1x TE. For each sample, 250 ng of its genomic DNA was randomly fragmented to 200-400 bp using NEBNext dsDNA Fragmentase (NEB). The fragmented DNA was purification with AMpure XP beads (Beckman Coulter). The purified DNA was used for Illumina library preparation with NEBNext Ultra DNA Library Prep Kit (New England Biolabs) (Fig. 1a). Each sample was labeled with a unique 8-bp index sequence. Three or four libraries were mixed into a pooled library in equal concentrations for subsequent hybridization capture.