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Methods for Generating Shotgun and Mixed Shotgun/Paired‐End Libraries for the 454 DNA Sequencer

Graham Wiley¹, Simone Macmil¹, Chunmei Qu¹, Ping Wang¹, Yanbo Xing¹, Doug White¹, Jianfeng Li¹, James D. White¹, Alexander Domingo², Bruce A. Roe¹

¹University of Oklahoma, Norman, Oklahoma
²Roche Diagnostics, Indianapolis, Indiana

Publication Name:

Current Protocols in Human Genetics

Unit Number:

Unit 18.1

DOI:

10.1002/0471142905.hg1801s61

Online Posting Date:

April, 2009

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Abstract

With the introduction of massively parallel, microminiature-based instrumentation for DNA sequencing, robust, reproducible, optimized methods are needed to prepare the target DNA for analysis using these high-throughput approaches because the cost per instrument run is orders of magnitude more than for typical Sanger dideoxynucleotide sequencing on fluorescence-based capillary systems. The methods provided by the manufacturer for genome sequencing using the 454/Roche GS-20 and GS-FLX instruments are robust. However, in an effort to streamline them for automation, we have incorporated several novel changes and deleted several extraneous steps. As a result of modifying these sample preparation protocols, the number of manual manipulations has also been minimized, and the overall yields have been improved for both shotgun and mixed shotgun/paired-end libraries. Curr. Protoc. Hum. Genet. 61:18.1.1-18.1.21. © 2009 by John Wiley & Sons, Inc.

Keywords: pyrosequencing; shotgun; paired-end; DNA Library

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Introduction
Basic Protocol 1: Shotgun Library Preparation
Basic Protocol 2: Shotgun/Paired-End Library Preparation
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Shotgun Library Preparation

Materials

Isopropanol, chilled to –80°C
GS DNA Library Preparation Kit (Roche), including:
- Nebulizer, with condenser and aspiration tubes
- TE buffer
- Nebulization buffer
- 10× polishing buffer
- BSA
- ATP
- dNTP mix
- T4 polynucleotide kinase
- T4 DNA polymerase
- 2× ligase buffer
- A and B adaptors
- Ligase
- Molecular biology grade water
- 10× Fill-In polymerase buffer
- Fill-In polymerase
3 to 5 µg purified, double-stranded DNA: e.g., see Roe et al. (1996), Roe (2004), appendix 3B, appendix 3C, or use a commercially available DNA isolation kit
Compressed nitrogen (research grade)
MinElute PCR Purification Kit (Qiagen), including:
- PBI buffer
- MinElute centrifuge columns
- PE buffer
- EB buffer
AMPure SPRI beads (Agencourt)
70% (v/v) ethanol

Dual-stage regulator on the nitrogen gas cylinder connected to the nebulizer using:
- ¼-in. (6.35-mm) hose barb fitting with ¼-in. (6.35-mm) NPT female compression fitting
- ¼-in. (6.35-mm) i.d. flexible tubing, with stainless steel screw clamp
1.5-ml microcentrifuge tubes
Microtiter plate for automated microfluidics system (Caliper)
Automated microfluidics system: AMS-90 and SE30 DNA LabChip (Caliper)
Magnetic particle concentrator (Invitrogen)
Vacuum drying system (e.g., model 5831; National Appliance)

NOTE: Most of the reagents in this protocol were obtained as components of the GS DNA Library Preparation Kit (Roche). The components of the kit are based on those described in the supplement to Margulies et al. (2005).

NOTE: All microcentrifugations are performed at room temperature.

Basic Protocol 2: Shotgun/Paired-End Library Preparation

Materials

3-5 µg purified, double-stranded DNA in TE buffer: e.g., see Roe et al. (1996), Roe (2004), appendix 3B, appendix 3C, or use a commercially available DNA isolation kit
TE buffer (appendix 2D)
AMPure SPRI beads (Agencourt)
70% (v/v) ethanol
MinElute PCR purification kit (Qiagen), including:
- EB buffer
- MinElute centrifuge columns
- PBI buffer
- PE buffer
32 mM S-adenosylmethionine (SAM) stock (New England Biolabs)
Molecular biology grade water
10× EcoRI methylase buffer and 40,000 U/ml EcoRI methylase (New England Biolabs)
10 mg/ml bovine serum albumin (BSA; New England Biolabs)
30,000 U/ml T4 polynucleotide kinase and 10× polynucleotide kinase buffer (USB)
10 mM (each) dNTP mix (see appendix 2D)
3000 U/ml T4 DNA polymerase (New England Biolabs)
2× Rapid Ligase buffer and Rapid Ligase (Roche)
Hairpin linker (GS DNA Paired End Adaptor Kit, Roche)
100 mM ATP (appendix 2D)
5000 U/ml lambda exonuclease (New England Biolabs)
10,000 U/ml T7 exonuclease (New England Biolabs)
5000 U/ml exonuclease I (New England Biolabs)
20,000 U/ml EcoRI
10× NEBuffer 4 (New England Biolabs)
Isopropanol, chilled to –80°C
GS DNA Library Preparation Kit (Roche), including:
- Nebulizer, with condenser and aspiration tubes
- TE buffer
- Nebulization buffer
- Polishing buffer
- BSA
- ATP
- dNTP mix
- T4 polynucleotide kinase
- T4 DNA polymerase
- 2× ligase buffer
- A and B adaptors
- Ligase
- Molecular biology grade water
- 10× Fill-In polymerase buffer
- Fill-In polymerase
Compressed nitrogen (research grade)

1.5-ml microcentrifuge tube
GeneMachines HydroShear apparatus, equipped with the~HydroShear large assembly and HydroShear syringe (Genomic Solutions)
Magnetic particle concentrator (Invitrogen)
Vacuum drying system (e.g., model 5831; National Appliance)
Microtiter plate for the automated microfluidics system (Caliper)
Automated microfluidics system: AMS-90 and SE30 DNA LabChip (Caliper)
Thermal cycler or heating block
12°C refrigerator
Dual-stage regulator on the nitrogen gas cylinder connected to the nebulizer using:
- ¼-in. (6.35-mm) hose barb fitting with ¼-in. (6.35-mm) NPT female compression fitting
- ¼-in. (6.35-mm) i.d. flexible tubing, with stainless steel screw clamp

NOTE: Most of the reagents used in this protocol were obtained as components of the GS DNA Library Preparation Kit (Roche). The components of the kit are based on those described in the supplement to Margulies et al. (2005).

NOTE: All microcentrifugations are performed at room temperature.

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Figures

Figure 18.1.1

Schematic representation of the shotgun and mixed shotgun,/paired-end library protocols. The left panel shows an outline of the steps needed to produce the shotgun library, which is subsequently quantitated, amplified by emPCR, and then sequenced on the 454/Roche GS-FLX sequencer. The right panel shows the steps used in the combined shotgun/paired-end protocol. Note that after the circularized DNA is nebulized, the end polishing, adaptor ligation, and fill-in is as shown in the shotgun protocol.

View Image
Figure 18.1.2

Results from a typical genomic DNA sample sheared with the Hydroshear and analyzed on a Caliper AMS-90.

View Image
Figure 18.1.3

Result from a typical combined shotgun/paired-end library analyzed on a Caliper AMS-90.

View Image

Literature Cited

Literature Cited
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	Roe, B. 2004. "Shotgun library construction for DNA sequencing" In Methods in Molecular Biology, vol 255: Bacterial Artificial Chromosomes, Volume 1: Library Construction, Physical Mapping, and Sequencing (S. Zhao and M. Stodolsky, eds.) pp. 171-187. Humana Press, Totowa, New Jersey.
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