MuSICA 2

MuSICA 2 reconstructs full-length cDNA clone sequences from Illumina Genome Analyzer 36-nucleotide short-read data to determine gene structures and alternative splice forms.

Key Features:

• Hybrid Assembly Approach: Combines de novo assembly with reference alignment of shotgun reads, where an external de novo assembler processes millions of short (36-nucleotide) reads from a single flow cell lane and the resulting assembly is refined by reference alignment.
• High Accuracy and Efficiency: In comparisons to primer-walking Sanger sequencing, it determined exon–intron structure for over 95% of human cDNA clones (excluding clones insufficiently represented due to PCR failure) and achieved nucleotide-level accuracy exceeding 99.99% for correctly assembled clones.
• Broad Applicability: Has been applied to full-length cDNA clones from non-human species, including Toxoplasma gondii, demonstrating cross-species utility.
• Cost-Effectiveness and Speed: The sequencing and assembly process can be completed in less than one week with consumables cost of approximately US$3 per clone.

Scientific Applications:

• Gene structure and alternative splicing analysis: Provides accurate full-length cDNA sequences for determining exon–intron structures and alternative splice forms.
• Protein-coding sequence characterization: Supplies complete coding sequences for experimental analyses of protein function.
• Large-scale cDNA sequencing projects: Enables rapid processing of numerous clones for high-throughput genomic studies.

Methodology:

Uses Illumina Genome Analyzer 36-nt short-read sequencing; performs de novo assembly of millions of short reads with an external assembler; refines the assembly by reference alignment of shotgun reads.