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.