Polony DNA Sequencing

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Polony DNA sequencing provides an inexpensive, accurate, high?throughput way to resequence genomes of interest by comparison to a reference genome. Mate?paired in vitro shotgun genomic libraries are produced and clonally amplified on microbeads by emulsion PCR. These serve as templates for sequencing by fluorescent nonamer ligation reactions on a microscope slide. Each sequencing run results in millions of 26?bp reads that can be aligned to the reference genome, allowing the identification of differences between sequences.

Keywords: DNA sequencing; high throughput; polony sequencing; resequencing; sequencing by synthesis

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Basic Protocol 1: Construction of a Shotgun Paired‐Tag Genomic Library
Basic Protocol 2: Emulsion PCR of Paired‐Tag Library on Microbeads
Basic Protocol 3: Enrichment for Amplicon‐Bearing Beads
Basic Protocol 4: Casting A Polony Bead Array
Basic Protocol 5: DNA Sequencing by Ligation With Degenerate Fluorescent Nonamers
Support Protocol 1: Titration of Template for Clonal Amplification by ePCR
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Construction of a Shotgun Paired‐Tag Genomic Library

Materials

Genomic DNA
Buffer EB (Qiagen)
End‐It DNA end repair kit (Epicentre), including:
- 10× buffer
- 10× ATP
- 10× dNTP mix
- Enzyme mix
10× PCR buffer without MgCl₂ (Invitrogen)
50 mM MgCl₂
100 mM dATP
5 U/µl Taq DNA polymerase
20 mg/ml glycogen
Oligonucleotides:
- 100 µM T30‐T: 5′‐phosphorylated‐GTCGGAGGCCAAGGCGGCCGTACGTCCAACT‐3′ (purified by HPLC)
- 100 µM T30‐B: 5′‐phosphorylated‐GTTGGACGTACGGCCGCCTTGGCCTCCGACT ‐3′ (purified by HPLC)
- 1 mM N6 oligonucleotides: 5′‐NNNundefinedundefinedN‐3′ (signifies_phosphorothioate_linkage~J_IDT~B~K~Hdiv~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~0~0~0~0~K~Hli~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~0~0~0~0~Kli~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~0~0~0~0~0~Kdiv~M~2~1~0~0~0~0~0~0~0~0~0~0~0~0~0~0~0~0~0~0~0100_µM FDV‐B: 5′‐ATCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGGTT‐3′ (purified by HPLC)
- 100 µM FDV‐T: 5′‐AACCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT‐3′ (purified by HPLC)
- 100 µM RDV‐B: 5′‐AGAGAATGAGGAACCCGGGGCAGTT‐3′ (purified by HPLC)
- 100 µM RDV‐T: 5′‐AACTGCCCCGGGTTCCTCATTCTCT‐3′ (purified by HPLC)
Quick ligation kit (NEB), including:
- 2× Quick ligation buffer
- Quick T4 DNA ligase
20 U/µl Exonuclease I (NEB)
100 U/µl Exonuclease III (NEB)
TE buffer, pH 8.0 ( appendix 22 )
25 mM dNTP mix (25 mM each nucleotide)
10× RepliPhi phi29 reaction buffer (Epicentre Technologies)
100 U/µl RepliPhi phi29 polymerase (Epicentre)
10× NEBuffer 4 (NEB)
1.6 mM S ‐adenosylmethionine (SAM; diluted from 32 mM stock in 1× NEBuffer 4; NEB)
2 U/µl Mme I (NEB)
2000 U/µl T4 DNA ligase and 10× buffer (NEB)
40% (w/v) polyethylene glycol 8000 (PEG) in H₂ O
10× NEBuffer 2 (NEB)
10 U/µl E. coli DNA polymerase I (NEB)
5 U/µl Platinum Taq DNA polymerase (Invitrogen)

Qiaquick columns (Qiaquick PCR cleanup kit; Qiagen)
NanoDrop ND‐1000 spectrophotometer (NanoDrop Technologies)
Microcon‐30 column (Millipore)
Thermal cycler

Additional reagents and equipment for DNA quantitation ( appendix 3D ), phenol/chloroform extraction and ethanol precipitation (unit 2.1 ), and agarose and polyacryamide gel electrophoresis (units 2.5 & 2.7 )

Basic Protocol 2: Emulsion PCR of Paired‐Tag Library on Microbeads

Materials

MyOne C1 1‐µm paramagnetic, streptavidin‐coated beads (Dynal)
Bind and wash (B&W) buffer (see recipe )
Oligonucleotides:
- 1 mM PR1‐F‐2BIO: 5′‐dual‐biotin‐CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT‐3′
- 2 mM PR1‐R: 5′‐CTGCCCCGGGTTCCTCATTCTCT‐3′
- 10 µM PR1‐3LF: 5′‐CCTCTCTATGGGCAGTCGGTGAT‐3′
TE buffer, pH 8.0 ( appendix 22 )
Light mineral oil (Sigma)
10% Span 80 (see recipe )
Tween 80
Triton X‐100
10× PCR buffer without MgCl₂ (Invitrogen)
50 mM MgCl₂
25 mM dNTP mix (25 mM each nucleotide)
5 U/µl Platinum Taq DNA polymerase (Invitrogen)
Template DNA (library DNA at appropriate concentration; see protocol 1 and protocol 6 )
NXS buffer (see recipe )
0.1 M NaOH

1.5‐ml microcentrifuge tubes
Magnetic particle concentrator (MPC; Dynal)
2‐ml cryogenic vials (Corning no. 430661)
Stir bar, flea‐size (VWR no. 58948‐353)
Magnetic stirrer (closed‐loop; VWR)
200‐µl eight‐tube PCR strips
Thermal cycler

Basic Protocol 3: Enrichment for Amplicon‐Bearing Beads

Materials

Spherotech particles (3‐µm streptavidin‐coated polystyrene particles; Spherotech no. SVP‐30‐5)
Bind and wash (B&W) buffer (see recipe )
1 mM PR1‐BIOXL: 5′‐biotinTEG‐CGTACCCCGCTTGGTCTTTCTCCCGTACCCCGCTTGGTCTTTCTCCCTGCCCCGGGTTCCTCATTCTCT‐3′
TE buffer, pH 8.0 ( appendix 22 )
ePCR beads ( protocol 2 )
Glycerol
0.1 M NaOH

Magnetic particle concentrator (MPC; Dynal)
1.5‐ml microcentrifuge tubes

Basic Protocol 4: Casting A Polony Bead Array

Materials

1% Triton X‐100 in H₂ O
Glacial acetic acid
Bind Silane (Promega)
100% ethanol
Enriched ePCR beads in TE buffer, pH 8.0 ( protocol 3 )
40% acrylamide/bisacrylamide (19:1) solution
5% TEMED
0.5% ammonium persulfate (APS)
Wash 1 (see recipe )

Round coverslips, 40 mm diameter, no. 1.5 (Bioptechs no. 40‐1313‐0319)
1000‐ml plastic beaker
Shaker
Vacuum desiccator
Teflon‐masked microscope slides (Erie Scientific, no. ER‐203W)

Basic Protocol 5: DNA Sequencing by Ligation With Degenerate Fluorescent Nonamers

Materials

Bead array with template DNA ( protocol 4 )
5× tailing buffer (Invitrogen)
1.25 mM ddNTP mix (1.25 mM each dideoxynucleotide)
15 U/µl terminal deoxytransferase (recombinant; Invitrogen)
Wash 1 (see recipe )
6× SSPE ( appendix 22 )/0.01% Triton X‐100
Anchor primer (see Fig. )
2000 U/µl T4 DNA ligase and 10× buffer (NEB)
100 µM degenerate fluorescent nonamer mix (Integrated DNA Technologies)
TE buffer, pH 8.0 ( appendix 22 )
1 U/µl USER enzyme mix (NEB)

Automated fluorescent microscope with flow cell (see Table 7.8.1 for parts list)
Autosampler (see Table 7.8.1 )
1‐ml disposable syringe
0.45‐µm pore size, 4‐mm cellulose acetate syringe filter (VWR)

Support Protocol 1: Titration of Template for Clonal Amplification by ePCR

Fluorescently labeled probes:
- 100 µM T30‐P2‐Cy5‐A: 5′‐Cy5‐AGUUGGACGUACGGCC‐3′
- 100 µM T30‐P2‐Cy5‐B: 5′‐Cy5‐AGUCGGAGGUCAAGGC‐3′
Heat block or slide thermal cycler
Microscope for bright‐field and fluorescence microscopy, with CCD camera and filter set for Cy5

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Figures

Figure 7.8.1 General procedure for polony sequencing. A paired‐tag genomic library is used as template for emulsion PCR on microbeads to generate polymerase colonies (polonies). The beads are cast on a coverslip in a thin layer of polyacrylamide, and put through iterative cycles of single‐base sequencing.

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Figure 7.8.2 Schematic of the 135‐bp bead‐bound template and sequences of oligonucleotides used in sequencing reactions (). For each reaction, the combination of anchor primer(s) and fluorescent nonamers is specified by the position in the tag being sequenced. All anchor primers should be synthesized with deoxyuridine to allow enzymatic removal of the primers. Fluorescent nonamers should be synthesized by Integrated DNA Technologies (IDT) with fully degenerate positions hand mixed to ensure equal ratios of each nucleotide at each position. The T30 sequence is shown as T30L and T30R to emphasize that the sequencing primers do not bind the full length of T30. Abbreviations: DMA and DMB, distal minus A and B strands; DP, distal plus; PM, proximal minus; PPA and PPB, proximal plus A and B strands; PR1‐F and PR1‐R, forward and reverse PR1 primers.

View Image

Videos

Literature Cited

Literature Cited
	Diehl, F., Li, M., Dressman, D., He, Y., Shen, D., Szabo, S., Diaz, L.A. Jr., Goodman, S.N., David, K.A., Juhl, H., Kinzler, K.W., and Vogelstein, B. 2005. Detection and quantification of mutations in the plasma of patients with colorectal tumors. Proc. Natl. Acad. Sci. U.S.A. 102:16368‐16373.
	Diehl, F., Li, M., He, Y., Kinsler, K.W., Vogelstein, B., and Dressman, D. 2006. BEAMing: Single‐molecule PCR on microparticles in water‐in‐oil emulsions. Nat. Methods 3:551‐559.
	Dressman, D., Yan, H., Traverso, G., Kinzler, K.W., and Vogelstein, B. 2003. Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proc. Natl. Acad. Sci. U.S.A. 100:8817‐8822.
	Li, M., Diehl, F., Dressman, D., Vogelstein, B., and Kinzler, K.W. 2006. BEAMing up for detection and quantification of rare sequence variants. Nat. Methods 3:95‐97.
	Mitra, R.D. and Church, G.M. 1999. In situ localized amplification and contact replication of many individual DNA molecules. Nucleic Acids Res. 37:e34.
	Mitra, R.D., Butty, V.L., Shendure, J., Benjamin, R., Williams, B.R., Housman, D.E., and Church, G.M. 2003. Digital genotyping and haplotyping with polymerase colonies. Proc. Natl. Acad. Sci. U.S.A. 100:5926‐5931.
	Sanger, F., Nicklen, S., and Coulson, A.R. 1977. DNA sequencing with chain‐terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74:5463‐5467.
	Shendure, J., Mitra, R.D., Varma, C., and Church, G.M. 2004. Advanced sequencing technologies: Methods and goals. Nat. Rev. Genet. 5:335‐344.
	Shendure, J., Porreca, G.J., Reppas, N.B., Lin, X., McCutcheon, J.P., Rosenbaum, A.M., Wang, M.D., Zhang, K., Mitra, R.D., and Church, G.M. 2005. Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309:1728‐1732.
	Siebert, P.D., Chenchik, A., Kellogg, D.E., Lukyanov, K.A., and Lukyanov, S.A. 1995. An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res. 23:1087‐1088.
	Turner, D.J., Shendure, J., Porreca, G., Church, G., Green, P., Tyler‐Smith, C., and Hurles, M.E. 2006. Assaying chromosomal inversions by single‐molecule haplotyping. Nat. Methods 3:439‐445.
	Zhang, K., Zhu, J., Shendure, J., Porreca, G.J., Aach, J.D., Mitra, R.D., and Church, G.M. 2006. Long‐range polony haplotyping of individual human chromosome molecules. Nat Genet. 38:382‐387.
	Zhu, J., Shendure, J., Mitra, R.D., and Church, G.M. 2003. Single molecule profiling of alternative pre‐mRNA splicing. Science 301:836‐838 .

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Polony DNA Sequencing

Abstract

Table of Contents

Materials

Basic Protocol 1: Construction of a Shotgun Paired‐Tag Genomic Library

Basic Protocol 2: Emulsion PCR of Paired‐Tag Library on Microbeads

Basic Protocol 3: Enrichment for Amplicon‐Bearing Beads

Basic Protocol 4: Casting A Polony Bead Array

Basic Protocol 5: DNA Sequencing by Ligation With Degenerate Fluorescent Nonamers

Support Protocol 1: Titration of Template for Clonal Amplification by ePCR

Figures

Videos

Literature Cited

关于丁香通

公司信息

个人用户

企业机构