amplikyzer

amplikyzer processes bisulfite flowgram sequencing data from the Roche 454 GS Junior to perform locus-specific DNA methylation analysis of amplicons by aligning flowgram intensity sequences to reference amplicon sequences.

Key Features:

• Direct alignment of intensity sequences: Aligns intensity sequences directly from standard flowgram files (SFF) to reference amplicon sequences without converting them to nucleotide FASTA.
• Preservation of flow intensities: Retains quantitative flow intensity information to avoid information loss caused by rounding during SFF-to-FASTA conversion.
• Handling of long T homopolymers: Applies specialized algorithms to process long T homopolymers in bisulfite-converted reads to mitigate over- and undercalls typical of pyrosequencing.
• Support for complex assay designs: Manages analyses involving multiple regions of interest (ROIs) and provides options for multiplexed sample analysis.
• Analytical outputs: Computes statistics and generates plots summarizing methylation analysis results.
• Hybrid representation: Utilizes a hybrid flowgram–DNA sequence representation underlying its alignment and analysis algorithms.

Scientific Applications:

• Locus-specific methylation analysis: Quantifies DNA methylation at specific amplicons derived from bisulfite treatment.
• Bisulfite flowgram sequencing on Roche 454 GS Junior: Processes datasets produced by the Roche 454 GS Junior pyrosequencing platform.
• Epigenetic research and diagnostics: Supports studies and diagnostic workflows that require accurate methylation calls despite homopolymer-associated sequencing errors.
• Multiplexed ROI studies: Enables analysis of complex, multiplexed assays spanning multiple regions of interest across samples.

Methodology:

Aligns intensity sequences from SFF flowgram files directly to reference amplicon sequences without conversion to FASTA, preserves flow intensities to avoid rounding-induced information loss, applies specialized algorithms to process long T homopolymers and pyrosequencing over-/undercalls, and employs a hybrid flowgram–DNA sequence representation.