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Exploring possible DNA structures in real-time polymerase kinetics using Pacific Biosciences sequencer data.

Sawaya S, Boocock J, Black MA, Gemmell NJ - BMC Bioinformatics (2015)

Bottom Line: We find pausing around the (CGG)n repeat that may indicate the presence of G-quadruplexes in some of the sequencer reads.The (CG)n repeat does not appear to cause polymerase pausing, but its kinetics signature nevertheless suggests the possibility that alternative nucleotide conformations may sometimes be present.The analyses presented here can be reproduced on any Pacific Biosciences kinetics data for any DNA pattern of interest using an R package that we have made publicly available.

View Article: PubMed Central - PubMed

Affiliation: Institute for Behavioral Genetics, University of Colorado, Boulder, USA. sterlingsawaya@gmail.com.

ABSTRACT

Background: Pausing of DNA polymerase can indicate the presence of a DNA structure that differs from the canonical double-helix. Here we detail a method to investigate how polymerase pausing in the Pacific Biosciences sequencer reads can be related to DNA sequences. The Pacific Biosciences sequencer uses optics to view a polymerase and its interaction with a single DNA molecule in real-time, offering a unique way to detect potential alternative DNA structures.

Results: We have developed a new way to examine polymerase kinetics data and relate it to the DNA sequence by using a wavelet transform of read information from the sequencer. We use this method to examine how polymerase kinetics are related to nucleotide base composition. We then examine tandem repeat sequences known for their ability to form different DNA structures: (CGG)n and (CG)n repeats which can, respectively, form G-quadruplex DNA and Z-DNA. We find pausing around the (CGG)n repeat that may indicate the presence of G-quadruplexes in some of the sequencer reads. The (CG)n repeat does not appear to cause polymerase pausing, but its kinetics signature nevertheless suggests the possibility that alternative nucleotide conformations may sometimes be present.

Conclusion: We discuss the implications of using our method to discover DNA sequences capable of forming alternative structures. The analyses presented here can be reproduced on any Pacific Biosciences kinetics data for any DNA pattern of interest using an R package that we have made publicly available.

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Correlations between wavelet coefficients. The Pearson’s correlation between all of the wavelet coefficients are shown for scales from 2 nucleotides to 2046 nucleotides. Red indicates a positive correlation, blue a negative correlation. The correlations between detail coefficients are on the bottom, and the correlations between smooth coefficients are on the top. The power for the detail coefficients is in the diagonal, indicating the relative variation at the different scales. The most easily interpreted results are on the top row, in which correlations shown here are similar to correlations between densities in a sliding window. The frequency of each base in these reads is 25%. Only reads longer than 1,000 nucleotides were used to avoid complications caused by boundaries between reads. A total of 223 nucleotides were examined (approximately 8.4 million nucleotides).
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Fig1: Correlations between wavelet coefficients. The Pearson’s correlation between all of the wavelet coefficients are shown for scales from 2 nucleotides to 2046 nucleotides. Red indicates a positive correlation, blue a negative correlation. The correlations between detail coefficients are on the bottom, and the correlations between smooth coefficients are on the top. The power for the detail coefficients is in the diagonal, indicating the relative variation at the different scales. The most easily interpreted results are on the top row, in which correlations shown here are similar to correlations between densities in a sliding window. The frequency of each base in these reads is 25%. Only reads longer than 1,000 nucleotides were used to avoid complications caused by boundaries between reads. A total of 223 nucleotides were examined (approximately 8.4 million nucleotides).

Mentions: Figure 1 shows the pairwise correlations between smooth wavelet coefficients (top right) and detail wavelet correlations (bottom left), with positive correlations highlighted in red and negative correlations highlighted in blue. The diagonal displays the relative power of the wavelet coefficients, indicating the strength of ability to test for correlations between the coefficients at that scale. For all of the coefficients examined here, the finest scales have the highest power.Figure 1


Exploring possible DNA structures in real-time polymerase kinetics using Pacific Biosciences sequencer data.

Sawaya S, Boocock J, Black MA, Gemmell NJ - BMC Bioinformatics (2015)

Correlations between wavelet coefficients. The Pearson’s correlation between all of the wavelet coefficients are shown for scales from 2 nucleotides to 2046 nucleotides. Red indicates a positive correlation, blue a negative correlation. The correlations between detail coefficients are on the bottom, and the correlations between smooth coefficients are on the top. The power for the detail coefficients is in the diagonal, indicating the relative variation at the different scales. The most easily interpreted results are on the top row, in which correlations shown here are similar to correlations between densities in a sliding window. The frequency of each base in these reads is 25%. Only reads longer than 1,000 nucleotides were used to avoid complications caused by boundaries between reads. A total of 223 nucleotides were examined (approximately 8.4 million nucleotides).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4384361&req=5

Fig1: Correlations between wavelet coefficients. The Pearson’s correlation between all of the wavelet coefficients are shown for scales from 2 nucleotides to 2046 nucleotides. Red indicates a positive correlation, blue a negative correlation. The correlations between detail coefficients are on the bottom, and the correlations between smooth coefficients are on the top. The power for the detail coefficients is in the diagonal, indicating the relative variation at the different scales. The most easily interpreted results are on the top row, in which correlations shown here are similar to correlations between densities in a sliding window. The frequency of each base in these reads is 25%. Only reads longer than 1,000 nucleotides were used to avoid complications caused by boundaries between reads. A total of 223 nucleotides were examined (approximately 8.4 million nucleotides).
Mentions: Figure 1 shows the pairwise correlations between smooth wavelet coefficients (top right) and detail wavelet correlations (bottom left), with positive correlations highlighted in red and negative correlations highlighted in blue. The diagonal displays the relative power of the wavelet coefficients, indicating the strength of ability to test for correlations between the coefficients at that scale. For all of the coefficients examined here, the finest scales have the highest power.Figure 1

Bottom Line: We find pausing around the (CGG)n repeat that may indicate the presence of G-quadruplexes in some of the sequencer reads.The (CG)n repeat does not appear to cause polymerase pausing, but its kinetics signature nevertheless suggests the possibility that alternative nucleotide conformations may sometimes be present.The analyses presented here can be reproduced on any Pacific Biosciences kinetics data for any DNA pattern of interest using an R package that we have made publicly available.

View Article: PubMed Central - PubMed

Affiliation: Institute for Behavioral Genetics, University of Colorado, Boulder, USA. sterlingsawaya@gmail.com.

ABSTRACT

Background: Pausing of DNA polymerase can indicate the presence of a DNA structure that differs from the canonical double-helix. Here we detail a method to investigate how polymerase pausing in the Pacific Biosciences sequencer reads can be related to DNA sequences. The Pacific Biosciences sequencer uses optics to view a polymerase and its interaction with a single DNA molecule in real-time, offering a unique way to detect potential alternative DNA structures.

Results: We have developed a new way to examine polymerase kinetics data and relate it to the DNA sequence by using a wavelet transform of read information from the sequencer. We use this method to examine how polymerase kinetics are related to nucleotide base composition. We then examine tandem repeat sequences known for their ability to form different DNA structures: (CGG)n and (CG)n repeats which can, respectively, form G-quadruplex DNA and Z-DNA. We find pausing around the (CGG)n repeat that may indicate the presence of G-quadruplexes in some of the sequencer reads. The (CG)n repeat does not appear to cause polymerase pausing, but its kinetics signature nevertheless suggests the possibility that alternative nucleotide conformations may sometimes be present.

Conclusion: We discuss the implications of using our method to discover DNA sequences capable of forming alternative structures. The analyses presented here can be reproduced on any Pacific Biosciences kinetics data for any DNA pattern of interest using an R package that we have made publicly available.

Show MeSH