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Open Access Methodology article

Enhanced 5-methylcytosine detection in single-molecule, real-time sequencing via Tet1 oxidation

Tyson A Clark1, Xingyu Lu2, Khai Luong1, Qing Dai2, Matthew Boitano1, Stephen W Turner1, Chuan He2 and Jonas Korlach1*

Author Affiliations

1 Pacific Biosciences, 1380 Willow Road, Menlo Park, CA 94025, USA

2 Department of Chemistry and Institute for Biophysical Dynamics, 929 East 57th Street, The University of Chicago, Chicago, Illinois, 60637, USA

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BMC Biology 2013, 11:4  doi:10.1186/1741-7007-11-4

Published: 22 January 2013

Additional files

Additional file 1:

Sequence context dependence of the kinetic signatures for 5mC and 5caC. Top panel (a) is a schematic of the synthetic SMRTbell template with random bases surrounding 5mC or 5caC modifications in a CG sequence context. The modified position is indicated with pink text and an asterisk. The bottom panel (b) is a heat map of IPD ratio values of either 5mC or 5caC relative to an unmodified control sequence. IPD ratio values are shown for all possible sequence contexts of four random bases over ten positions on the DNA template (-3 to +6 relative to the modified base). Light grey boxes within the heatmap denote sequence contexts that did not have sufficient sequencing coverage. A minimum of 10 independent molecules of both modified and control templates were analyzed.

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Open Data

Additional file 2:

Conversion of 5mC to 5caC in synthetic oligonucleotides. Kinetic signals for synthetic oligonucleotides carrying two 5mC modified sites (red bars) are shown before (top) and after (bottom) mTet1-mediated oxidation to 5caC. IPD ratio data are plotted for each template position relative to a control template of identical sequence but lacking modifications. The template is shown in the 5' to 3' direction from left to right, the polymerase movement is right to left across the template as indicated by the arrow.

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Open Data

Additional file 3:

IPD ratio distributions of all methylated motifs in E.coli MG1655 and B.halodurans C-125. Each plots show the histograms of IPD ratio values for each methylated motif and an off-target non-methylated motif. The top plots are from native samples and the bottom show the same data after Tet1-mediated conversion of 5mC to 5caC.

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Open Data

Additional file 4:

Table of detection rates for all methylated motifs in E.coli MG1655. The number and percent detection is shown for all methylated sequence motifs in the genome. A detected genomic position is one that has a kinetic score that is greater than the cutoff value. Detection rates are also shown for common secondary IPD ratio peaks of 6mA (+5) and 5mC (+2, +6) and for off-target motifs with similar sequence content to the methylated motifs. Methylated bases are colored: 6mA (red), 5mC (blue). The interrogated base in the motif is underlined. Unassigned are genomic positions with kinetic scores above the cutoff which are not in a methylated motif or a secondary peak.

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Open Data

Additional file 5:

Table of detection rates for all methylated motifs in B.halodurans C-125. The number and percent detection is shown for all methylated sequence motifs in the genome. A detected genomic position is one that has a kinetic score that is greater than the cutoff value. Detection rates are also shown for common secondary IPD ratio peaks of 6mA (+5) and 5mC (+2, +6) and for off-target motifs with similar sequence content to the methylated motifs. Methylated bases are colored: 6mA (red), 5mC (blue). The interrogated base in the motif is underlined. Unassigned are genomic positions with kinetic scores above the cutoff which are not in a methylated motif or a secondary peak.

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Open Data

Additional file 6:

Table of oligonucleotide sequences used in this study.

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Open Data