Construction and sequencing of DNA libraries on Hiseq 2000 platform for the eastern banjo frog
Qiye Li, Qunfei Guo, Yang Zhou, Huishuang Tan, Terry Bertozzi, Yuanzhen Zhu, Ji Li, Stephen Donnellan, Guojie Zhang
Abstract
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
The following step is the protocol for construction and sequencing of short-insert libraries (170 bp, 250 bp, 500 bp, and 800 bp).
Genomic DNA interruption
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).
End-repair
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.
0h 30m 0s
20°C
Add adapter
T-tailed adapters were ligated to both ends of these DNA fragments and amplified.
PCR amplification
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.
0h 3m 0s
95°C
0h 0m 20s
98°C
0h 0m 15s
60°C
72°C
0h 10m 0s
72°C
Library purification
AMPure XP beads (Agencourt, Beverly, the U.S.) was used to purify the PCR production.
Sequencing
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).
The following step is the protocol for construction and sequencing of mate-paired libraries (2 kb, 5 kb, 10 kb, and 20 kb).
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).
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).
Repair by using T4 DNA polymerase (ENZYMATIC, Beverly, the U.S.) 30 min at 20 ℃.
0h 30m 0s
20°C
Biotin Label
Add Biotin Label by Biotin dNTP Mix (5mM) 30 min at 20 ℃.
0h 30m 0s
20°C
Fragment selection
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.
Fragment cyclizing
The T3 DNA ligase was used to connect the ring. And then, Covaris LE220 was used to cyclize DNA fragments.
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.).
0h 30m 0s
20°C
0h 0m 15s
0h 30m 0s
37°C
0h 0m 15s
Adaptor ligation (1h at 20 °C, 1000rmp vibrate for 15 sec per 2 min.).
1h 0m 0s
20°C
0h 0m 15s
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
0h 3m 0s
98°C
0h 0m 20s
60°C
0h 0m 15s
72°C
0h 0m 45s
72°C
0h 10m 0s
4°C
AMPure XP beads (Agencourt, Beverly, the U.S.) was used to purify the PCR production.
