Computational Systems Biology Methods and Protocols.7z

11. Separate hybridized target molecules from the bait in 30μLTE
    by incubation at 95C for 5 min in a thermal cycler.


12. The PCR condition is the same withstep6in Subheading3.2
    for capture hybridization.

3.4 Standard Library
Preparation

1. Shear the enriched libraries for 120 s using an IonShear kit
   (Thermo Fisher) in an open thermocycler. The 2 kb DNA
   fragments will be sheared to 300–500 bp.


2. Adaptor ligation is the same withstep4in Subheading3.2 for
   capture hybridization.


3. Select 450–500 bp reads by using 2% E-gel.


4. Library amplification is carried out in a PCR volume of 50μL
   by using a Library Amplification Kit:
   (a) 25μL HiFi mix.
   (b) 21μL selected fragment solution.
   (c) 4μL primer mix (from Ion Plus Fragment Library Kit,
   Thermo Fisher).


5. Concentration is measured by using Qubit. Length of library is
   measured by using 2100 Bioanalyzer (Agilent).

4 A Case for Capturing Mitochondrial Genome from Amphibia

Three samples were selected to illustrate the performance of

LR-LCR and standard capture hybridization method, including

Ranasp.1,Ranasp.2, andOnychodactylussp. To get all the three

mitogenomes as reference, we amplified them by using the primers

in Table1 and sequenced LR-PCR products directly. To get probe,

we use LR-PCR products fromRanasp.1 and total DNAs from

other two species to prepare libraries. DNA fromRanasp.2 and

Onychodactylussp. are separated into two parts, respectively. One

was used to construct libraries with length of 500 bp and another

with length of 2 kb. The length of 500 bp library was prepared by

using standard capture hybridization method; the length of 2 kb

library according to LR-LCR strategy was processed and

sequenced. All the libraries were listed in Table2.

We used the probe ofRanasp.1 to capture a closely related

mtDNA fromRanasp.2 (CO1K2P¼8.2%) and a relatively distant

mtDNA fromOnychodactylussp. (CO1K2P¼25.5%). Standard

capture hybridization and LR-LCH yielded an average depth per

base of 48.65 and 156.15 coverage forRanasp.2 andOnychodac-

tylussp., respectively (Table2). On the one hand, to evaluate the

capture sensitivity and specificity for standard capture hybridization

method, we observed that two gaps existed in the MtG ofRana

sp.2 at the end ofND5and in the noncoding regions. InOnycho-

dactylussp.1, five gaps occurred in relatively divergent regions at

38 Xing Chen et al.