Mapping Bisulfite-Treated Short DNA Reads

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Virginia Tech


Epigenetics are stable heritable traits that are not a result of the DNA sequence. Epigenetic modification of DNA cytosine plays a role in development and disease. The covalent bonding of a methyl group or a hydroxymethyl group to the 5-carbon of cytosine epigenetically modifies cytosine to 5-methylcytosine or 5-hydroxymethylcytosine. Upon PCR amplification, the bisulfite treatment of DNA converts unmethylated cytosine to thymine, while 5-methylcytosine, 5-hydroxymethylcytosine, and other bases remain unchanged. The resulting sequences can be mapped to a reference genome; however, this can be challenging due to sequencing technology complexity, low sequence complexity, and biases and errors introduced with bisulfite treatment. Once the short read is mapped, the identity of 5-methylcytosine or 5-hydroxymethylcytosine can be determined by comparing the mapped read to the aligned reference genome. Bisulfite DNA read mapping is characterized by mapping performance as low as 40%. This research improves bisulfite short read mapping quality. First, reads generated from the bisulfite hairpin PCR protocol are used to study mapping failure and solutions. A read may not map to the genome; it may map uniquely, or it may map to multiple locations. Sequence complexity correlates with these mapping categories. The hairpin protocol allows for a recovery, in some cases, of the original untreated read, and mapping this read with the regular read mapper Bowtie2 improved mapper performance by 10%. New bisulfite read mapping software called BisPin was created that calls BFAST (BLAT-like Fast Accurate Search Tool) for mapping. BisPin resolves ambiguously mapped reads with a rescoring strategy, which yields a statistically significant improvement. BFAST-Gap for Ion Torrent reads was developed, since Ion Torrent machines are less expensive than Illumina machines and since Ion Torrent reads are longer. There are few mappers for Ion Torrent data. BFAST-Gap uses homopolymer run length for contextual gap penalty functions, since homopolymer runs cause errors in Ion Torrent reads. In conjunction with BisPin, this software performed well on real and simulated bisulfite Ion Torrent data and Illumina data. InfoTrim, a read trimmer with an entropy term, was developed with competitive results.



DNA read alignment, hairpin whole genome bisulfite, indels, bisulfite Ion Torrent, BisPin, BFAST-Gap