Some studies provided solutions for increasing probe capture
ability or reducing sequence distance between probes and target
DNA. Mason et al. utilized PCR product to make probe from a
single extant species Sunda colugo (Galeopterus variegatus). The
probes successfully capture up to 13% divergence of target mito-
chondrial DNA (mtDNA), on average 76.92% capture specificity,
and on average 56.29% capture sensitivity for 13 museum speci-
mens of Sunda colugo [1]. Penalba et al. used long-range PCR
(LR-PCR) product to make probe, and its capture efficiency
increased rapidly for 27% divergence of target mtDNA from lizards
[21]. Li et al. tested the various hybridization temperatures, touch-
down strategy, and second-bait strategy for five pairs of animal
groups (up to 298.6 Mya for western clawed toad). The study
shows improvement of capture specificity to some degree [22]. Paij-
mans et al. examined the various temperatures, such as standard
hybridization temperature 65C and low temperatures 60, 50,
48, and 45 C, and touchdown strategy for both fresh and
degraded DNA from carnivoran family Felidae. They concluded
that capture specificity improved when applying 65C for degraded
samples and touchdown strategy for fresh samples. There is no
effect on improving capture sensitivity by adjusting hybridization,
suggesting the temperature is not a crucial parameter to get diver-
gent DNA [23].
Based on the observation of previous studies for divergent
distance analysis across entire mitochondrial genome (mitogen-
ome) and chromosome from animals, we noticed that a gene
usually consists of relatively conservative and divergent parts. In
other words, not all parts in a long locus have constant divergence.
The distributional pattern of divergent parts intercepted by the
conservative parts, despite the length of the former, was variable
from one to another. For example, Mason et al. plotted a DNA
sequence identity picture across the complete mitogenomes for
Sunda colugo. Divergent part in conservative gene16sRNA is in
the middle part and the beginning ofCO1is much more divergent.
Conservative region of divergent genes ND1,ND2, andND5
located in the middle part and for control region is at its beginning
(Fig. 4c in ref.1). Li et al. plotted a phylogenetic signal density
picture across entire chromosome A1 and chromosome X for leop-
ard (Pantheragenus). These divergent and conservative parts were
alternatively distributed in chromosome A1. Almost all the regions
in chromosome X matched the pattern with some exceptions near
the regions about 10, 25, 90, and 180 Mb (Fig. 4d in ref.24).
In this study, to address the problem of capturing divergent
DNA, we propose to modify a library by extending it to a longer
length during the step 2 described above. We hypothesize that
divergent DNA can be enriched along with its adjacent conservative
DNA which can be captured easily (hereafter termed this strategy as
LR-LCH). The general pipeline is shown in Fig.1. First, we32 Xing Chen et al.