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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.

Show MeSH
Kinetics around CG repeats. These three graphs show the average polymerase kinetics for the sequence CGCGCGCGCG, found in 18 regions in the E. coli genome. A total of 746 reads were used in the analysis. The top graph shows the raw IPD values for the region, in seconds. In the middle and bottom graphs, the 2 bp (4 bp) smoothing represents wavelet smoothing over a two (four) nucleotide region within the reads. For each measurement, the black vertical lines are the 90% quantile, the grey vertical lines are the 95% quantile.
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Fig5: Kinetics around CG repeats. These three graphs show the average polymerase kinetics for the sequence CGCGCGCGCG, found in 18 regions in the E. coli genome. A total of 746 reads were used in the analysis. The top graph shows the raw IPD values for the region, in seconds. In the middle and bottom graphs, the 2 bp (4 bp) smoothing represents wavelet smoothing over a two (four) nucleotide region within the reads. For each measurement, the black vertical lines are the 90% quantile, the grey vertical lines are the 95% quantile.

Mentions: The kinetics within this repeat produce an interesting pattern (Figure 5). Most of the cytosines in the repeat region have a slightly high average IPD value, while the guanines have a slightly lower average IPD value. More interesting, the cytosines can sometimes have an IPD over 1 second. As indicated by the vertical bars in Figure 5, 10% of the IPD values at most of the cytosines are over 1 second, and 5% are greater than approximately 2 seconds in duration. In contrast, the majority of the IPD values at the guanines are very small (most of their 90% and 95% ranges are less than 0.5 seconds).Figure 5


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)

Kinetics around CG repeats. These three graphs show the average polymerase kinetics for the sequence CGCGCGCGCG, found in 18 regions in the E. coli genome. A total of 746 reads were used in the analysis. The top graph shows the raw IPD values for the region, in seconds. In the middle and bottom graphs, the 2 bp (4 bp) smoothing represents wavelet smoothing over a two (four) nucleotide region within the reads. For each measurement, the black vertical lines are the 90% quantile, the grey vertical lines are the 95% quantile.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Kinetics around CG repeats. These three graphs show the average polymerase kinetics for the sequence CGCGCGCGCG, found in 18 regions in the E. coli genome. A total of 746 reads were used in the analysis. The top graph shows the raw IPD values for the region, in seconds. In the middle and bottom graphs, the 2 bp (4 bp) smoothing represents wavelet smoothing over a two (four) nucleotide region within the reads. For each measurement, the black vertical lines are the 90% quantile, the grey vertical lines are the 95% quantile.
Mentions: The kinetics within this repeat produce an interesting pattern (Figure 5). Most of the cytosines in the repeat region have a slightly high average IPD value, while the guanines have a slightly lower average IPD value. More interesting, the cytosines can sometimes have an IPD over 1 second. As indicated by the vertical bars in Figure 5, 10% of the IPD values at most of the cytosines are over 1 second, and 5% are greater than approximately 2 seconds in duration. In contrast, the majority of the IPD values at the guanines are very small (most of their 90% and 95% ranges are less than 0.5 seconds).Figure 5

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