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Decoding long nanopore sequencing reads of natural DNA.

Laszlo AH, Derrington IM, Ross BC, Brinkerhoff H, Adey A, Nova IC, Craig JM, Langford KW, Samson JM, Daza R, Doering K, Shendure J, Gundlach JH - Nat. Biotechnol. (2014)

Bottom Line: As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers).This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome.This work provides a foundation for nanopore sequencing of long, natural DNA strands.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, University of Washington, Seattle, Washington, USA.

ABSTRACT
Nanopore sequencing of DNA is a single-molecule technique that may achieve long reads, low cost and high speed with minimal sample preparation and instrumentation. Here, we build on recent progress with respect to nanopore resolution and DNA control to interpret the procession of ion current levels observed during the translocation of DNA through the pore MspA. As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers). This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome. Furthermore, we show nanopore sequencing reads of phi X 174 up to 4,500 bases in length, which can be unambiguously aligned to the phi X 174 reference genome, and demonstrate proof-of-concept utility with respect to hybrid genome assembly and polymorphism detection. This work provides a foundation for nanopore sequencing of long, natural DNA strands.

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Experimental schematic and raw data. (a) Method of adapting dsDNA for nanopore sequencing. The first adaptor (orange) includes a cholesterol tail which inserts into the membrane, increasing DNA capture rates32 while, the long 5' single stranded overhang facilitates insertion into the pore. A second adaptor (green) enables re-reading of the pore using the DNAP's synthesis mode11, 17. (b) The protein nanopore MspA is shown in blue, phi 29 DNAP in green and DNA in orange. An applied voltage across the bilayer drives an ion current through the pore and an amplifier measures the current. DNA bases within the constriction determine the ion current. Phi 29 DNAP steps DNA through the pore in single-nucleotide steps. (c–e) Raw data for a representative 3000-second time window. Ion current changes as DNA is fed through the pore in single-nucleotide steps. Panels d and e each show a 1% section of the preceding panel's data shaded in red.
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Figure 1: Experimental schematic and raw data. (a) Method of adapting dsDNA for nanopore sequencing. The first adaptor (orange) includes a cholesterol tail which inserts into the membrane, increasing DNA capture rates32 while, the long 5' single stranded overhang facilitates insertion into the pore. A second adaptor (green) enables re-reading of the pore using the DNAP's synthesis mode11, 17. (b) The protein nanopore MspA is shown in blue, phi 29 DNAP in green and DNA in orange. An applied voltage across the bilayer drives an ion current through the pore and an amplifier measures the current. DNA bases within the constriction determine the ion current. Phi 29 DNAP steps DNA through the pore in single-nucleotide steps. (c–e) Raw data for a representative 3000-second time window. Ion current changes as DNA is fed through the pore in single-nucleotide steps. Panels d and e each show a 1% section of the preceding panel's data shaded in red.

Mentions: In nanopore devices directed at DNA sequencing, a salt solution is divided into cis and trans wells by a thin membrane. A single nanometer-scale pore in the membrane connects the cis and trans wells electrically. When a voltage is applied across this membrane, ion current flows through the pore; this current provides the primary signal. DNA is negatively charged and is electrophoretically attracted into the pore. When single-stranded (ss) DNA enters the pore, it blocks some fraction of the ion current. The fraction of the ion current that is blocked depends on the identity of nucleotides within the pore13–15. Key challenges of this technique are single-nucleotide resolution and control of the DNA translocation. Single-nucleotide resolution was recently enabled through the development of MspA, a protein pore with a short and narrow constriction11, 13, 15, 16. DNA translocation control was also recently enabled through the use of molecular motors such as phi29 DNA Polymerase (DNAP)11, 17 (Fig. 1).


Decoding long nanopore sequencing reads of natural DNA.

Laszlo AH, Derrington IM, Ross BC, Brinkerhoff H, Adey A, Nova IC, Craig JM, Langford KW, Samson JM, Daza R, Doering K, Shendure J, Gundlach JH - Nat. Biotechnol. (2014)

Experimental schematic and raw data. (a) Method of adapting dsDNA for nanopore sequencing. The first adaptor (orange) includes a cholesterol tail which inserts into the membrane, increasing DNA capture rates32 while, the long 5' single stranded overhang facilitates insertion into the pore. A second adaptor (green) enables re-reading of the pore using the DNAP's synthesis mode11, 17. (b) The protein nanopore MspA is shown in blue, phi 29 DNAP in green and DNA in orange. An applied voltage across the bilayer drives an ion current through the pore and an amplifier measures the current. DNA bases within the constriction determine the ion current. Phi 29 DNAP steps DNA through the pore in single-nucleotide steps. (c–e) Raw data for a representative 3000-second time window. Ion current changes as DNA is fed through the pore in single-nucleotide steps. Panels d and e each show a 1% section of the preceding panel's data shaded in red.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4126851&req=5

Figure 1: Experimental schematic and raw data. (a) Method of adapting dsDNA for nanopore sequencing. The first adaptor (orange) includes a cholesterol tail which inserts into the membrane, increasing DNA capture rates32 while, the long 5' single stranded overhang facilitates insertion into the pore. A second adaptor (green) enables re-reading of the pore using the DNAP's synthesis mode11, 17. (b) The protein nanopore MspA is shown in blue, phi 29 DNAP in green and DNA in orange. An applied voltage across the bilayer drives an ion current through the pore and an amplifier measures the current. DNA bases within the constriction determine the ion current. Phi 29 DNAP steps DNA through the pore in single-nucleotide steps. (c–e) Raw data for a representative 3000-second time window. Ion current changes as DNA is fed through the pore in single-nucleotide steps. Panels d and e each show a 1% section of the preceding panel's data shaded in red.
Mentions: In nanopore devices directed at DNA sequencing, a salt solution is divided into cis and trans wells by a thin membrane. A single nanometer-scale pore in the membrane connects the cis and trans wells electrically. When a voltage is applied across this membrane, ion current flows through the pore; this current provides the primary signal. DNA is negatively charged and is electrophoretically attracted into the pore. When single-stranded (ss) DNA enters the pore, it blocks some fraction of the ion current. The fraction of the ion current that is blocked depends on the identity of nucleotides within the pore13–15. Key challenges of this technique are single-nucleotide resolution and control of the DNA translocation. Single-nucleotide resolution was recently enabled through the development of MspA, a protein pore with a short and narrow constriction11, 13, 15, 16. DNA translocation control was also recently enabled through the use of molecular motors such as phi29 DNA Polymerase (DNAP)11, 17 (Fig. 1).

Bottom Line: As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers).This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome.This work provides a foundation for nanopore sequencing of long, natural DNA strands.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, University of Washington, Seattle, Washington, USA.

ABSTRACT
Nanopore sequencing of DNA is a single-molecule technique that may achieve long reads, low cost and high speed with minimal sample preparation and instrumentation. Here, we build on recent progress with respect to nanopore resolution and DNA control to interpret the procession of ion current levels observed during the translocation of DNA through the pore MspA. As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers). This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome. Furthermore, we show nanopore sequencing reads of phi X 174 up to 4,500 bases in length, which can be unambiguously aligned to the phi X 174 reference genome, and demonstrate proof-of-concept utility with respect to hybrid genome assembly and polymorphism detection. This work provides a foundation for nanopore sequencing of long, natural DNA strands.

Show MeSH
Related in: MedlinePlus