TITLE: Construction and sequencing of DNA libraries on Hiseq 2000 platform for the eastern banjo frog
AUTHORS: Qiye Li, Qunfei Guo, Yang Zhou, Huishuang Tan, Terry Bertozzi, Yuanzhen Zhu, Ji Li, Stephen Donnellan, Guojie Zhang

[DESCRIPTION]
This protocol is used  for construction and sequencing of DNA libraries which include short-insert libraries and mate-paired libraries on the Hiseq 2000 platform for the eastern banjo frog. 

[STEPS]
1. The following step is the protocol for construction and sequencing of short-insert libraries (170 bp, 250 bp, 500 bp, and 800 bp).

2. The following step is the protocol for construction and sequencing of mate-paired libraries (2 kb, 5 kb, 10 kb, and 20 kb).
SECTION: Genomic DNA interruption
1.1. The extracted 1 µg genomic DNA was randomly fragmented by Covaris E220 ultrasonicator (Covaris, Brighton, UK) to obtain ~170 bp, ~250 bp, ~500 bp, and ~800 bp fragments (for 170 bp, 250 bp, 500 bp, and 800 bp libraries respectively).
SECTION: End-repair
1.2. Repair by using T4 DNA polymerase (ENZYMATICS, Beverly, the U.S.) 30 min. at 20 ℃ to obtain blunt ends which were then 3’-adenlyated to create sticky ends.

 
30 min 
20 °C 
SECTION: Add adapter
1.3. T-tailed adapters were ligated to both ends of these DNA fragments and amplified.
SECTION: Sequencing
1.6. After purification, the library was qualified by the Agilent Technologies 2100 bioanalyzer and ABI StepOnePlus Realtime PCR System.
Finally, the qualified libraries were sequenced paired-end using Hiseq System (Illumina).
SECTION: Genomic DNA interruption
2.1. The genomic DNA was fragmented using a Covaris E220 ultrasonicator (Covaris, Brighton, UK) to obtain ~2 kb (for 2 kb library) and a Hydroshear (GeneMachines, CA, USA) to obtain ~5 kb, ~10 kb, ~20 kb fragments (for 5 kb, 10 kb, and 20 kb libraries respectively).

SECTION: End-repair
2.2. Repair by using T4 DNA polymerase (ENZYMATIC, Beverly, the U.S.) 30 min at 20 ℃.


30 min 
20 °C 

SECTION: Biotin Label
2.3. Add Biotin Label by Biotin dNTP Mix (5mM) 30 min at 20 ℃.

 
30 min 
20 °C 

SECTION: Fragment selection
2.4. These fragments were further selected into size ranges of 2–2.4 kb, 5–5.5 kb, 10–11 kb or 20-23 kb by agarose gel electrophoresis.
SECTION: Fragment cyclizing
2.5. The T3 DNA ligase was used to connect the ring. And then, Covaris LE220 was used to cyclize DNA fragments.

 

SECTION: End-repair
2.6. Fragmented DNA labeled with biotin was captured on M280 streptavidin beads (Invitrogen, CA, USA), followed by end repair (30 min. at 20°C, 1000 rotation per minute, rpm, vibrate for 15 sec. per 2 min.), A-tailing (30 min. at 37°C, 1000 rpm vibrate for 15 sec. per 2 min.).

 
30 min 
20 °C 
15 s 
30 min 
37 °C 
15 s 

SECTION: Add adapter
2.7. Adaptor ligation (1h at 20 °C, 1000rmp vibrate for 15 sec per 2 min.).
60 min 
20 °C 
15 s 

SECTION: PCR amplification
2.8. PCR amplifications on beads 95°C 3 min., (98 °C 20 sec., 60 °C 15 sec., 72 °C 45 sec.) for N (For 2 kb library, N=16; For 5 kb library, 10 kb library and 20 kb library, N=18) cycles, 72 °C 10 min., 4°C hold using Enzymatics (MA, USA) and NEB (MA, USA) reagent.
95 °C 
3 min 
98 °C 
20 s 
60 °C 
15 s 
72 °C 
45 s 
72 °C 
10 min 
4 °C 

SECTION: Sequencing
2.10. After purification, the library was qualified by the Agilent Technologies 2100 bioanalyzer and ABI StepOnePlus Realtime PCR System.
Finally, the qualified libraries were sequenced paired-end using Hiseq System (Illumina).
SECTION: PCR amplification
1.4. The temperature profile was 3 min. at 95 ℃ followed by 8 cycles of 20 sec. at 98 ℃, 15 sec. at 60 ℃, 30 sec. at 72 ℃, and more 10 min. at 72 ℃ for further elongation.
3 min 
95 °C 
20 s 
98 °C 
15 s 
60 °C 
  
72 °C 
10 min 
72 °C 

SECTION: Library purification
1.5. AMPure XP beads (Agencourt, Beverly, the U.S.) was used to purify the PCR production.
SECTION: Library purification
2.9. AMPure XP beads (Agencourt, Beverly, the U.S.) was used to purify the PCR production.