(2) prepare RNA/DNA library; (3) mix the probe and library to
hybridize; (4) sequence hybridized library fragments. As compared
to the old protocol, adding the second step has two obvious merits.
The first is the copy number of target DNA from low level to
moderate or high level. This results in great improvement of
probe capture ability. The final process of library construction in
step 2 is amplify the adapter-ligated DNA within 18 cycles. The
quantity of library rapidly increased about 100–1000 times (from
less than 1 ng to about 100–1000 ng depended on the sample
quality). Tsangaras et al. demonstrated that the reduction of quan-
tity of DNA library from 1500 to 15 ng results in the rapid decrease
of rate of on-target reads in raw data (termed capture specificity)
and the coverage rate of target genome region (termed capture
sensitivity, [18]). Second, shorter than 500 bp of library length has
been considered the best choice for both capture hybridization and
sequencing experiments in order to get the best capture specificity
and sensitivity. Gnirke et al. suggested that in-solution hybridiza-
tion may be more efficient for libraries with fragment shorter than
500 bp [12, 19]. This length of libraries facilitated the most popu-
lar HTS platforms, such as less than 650 bp for MiSeq, less than
350 bp for HiSeq, and less than 480 bp for Ion Torrent PGM (the
length included adaptor and index for these sequence machines).
But when using single molecular sequence technology, the length
of 500 bp was no longer a best parameter setting anymore since it
enables sequencing the DNA fragment larger than 5 kb. Karamitros
and Magiorkinis examined the efficiency when using 5 and 10 kb
libraries for two long loci of interest fromphage lambdaandEscher-
ichia coliand followed sequencing by using Oxford Nanopore
MinION. The efficiency of their method is very well with 92.5%
capture specificity and 99.73% capture sensitivity [20].
Researchers always require to balance the probe accessibility
and sequence distance between probes and target DNA problem in
order to increase capture sensitivity and capture specificity as high
as possible [20–22]. There are some studies that examined the
capture ability of various capture hybridization methods for diver-
gent DNA sequence. Hedtke et al. designed an exon probe refer-
ring the reference genome of western clawed frog (Xenopus
tropicalis) to capture other 16 frog samples (diversification dates
to about 250 Mya). There is a negative correlation between diver-
gence time and the number of on-target reads [10]. Bi et al.
designed probe by referring the de novo assembled transcriptome
contigs from the alpine chipmunk (Tamias alpinus). Within 1.5%
sequence divergence in coding regions, there is no decline of cap-
ture specificity (24.4–29.1%) and capture sensitivity (about 90%).
But both parameters decreased in the more divergent loci (about
9% sequence diversity, 30 Mya) from another genome of squirrel
(Ictidomys tridecemlineatus,[15]).Capture Hybridization of Long-Range DNA Fragments for High-Throughput Sequencing 31