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

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Protocol publication

[…] SMRTbell templates were subjected to standard SMRT sequencing, as described [,]. Reads were processed and mapped to the respective reference sequences using the BLASR mapper [] and Pacific Biosciences' SMRT Analysis pipeline [] using the standard mapping protocol. IPDs were measured as previously described [] and processed as described [] for all pulses aligned to each position in the reference sequence.For the bacterial methylome analysis [], we used Pacific Biosciences' SMRTPortal analysis platform v. 1.3.1, which uses an in silico kinetic reference and a t-test based detection of modified base positions []. The following GenBank reference sequences were used: U00096.2 for E. coli K-12 MG1655 and BA000004.3 for B. halodurans C-125. MTase target sequence motifs were identified by selecting the top 1,000 kinetic hits and subjecting a ±20 base window around the detected base to MEME-ChIP [], and compared to the predictions in REBASE []. To estimate the enhancement of detection of methylated 5mC positions (Table ), we first selected an orthogonal off-target motif of similar sequence content and calculated the kinetic score representing the 99th percentile of all genomic positions of that motif (5'-GGWCC-3' for E. coli (score threshold = 35.6); 5'-CCGG-3' for B. halodurans (30.4)). We then used this 1% false positive detection threshold for determining the number of genomic positions of the on-target methylation sites detected as methylated (Figures and ; Table ). IPD ratio plots were visualized using Circos []. […]

Pipeline specifications

Software tools BLASR, SMRT-Analysis, MEME-ChIP, Circos
Databases REBASE
Application Genome data visualization
Organisms Escherichia coli str. K-12 substr. MG1655
Chemicals Cytosine, 5-Methylcytosine