MuSICA 2: Multi-clone Shotgun Integrated cDNA Assembler 2 jump to the main text


Sequencing full-length cDNA clones is the most robust way to annotate genes. Recently, with the advent of new-generation sequencing instruments such as Illumina Genome Analyzer (Illumina GA) and AppliedBiosystem SOLiD, genome-wide comprehensive approaches including RNA-sequencing succeeded in capturing both expressed regions and expression levels of genes without cloning individual mRNAs. However, RNA expression level varies widely at least four orders of magnitude, and thus it is impossible to robustly capture lowly expressed transcripts without cloning or other nomalization techniques. Moreover, overlapping transcripts (alternative splicing variants, alternative transcription start/end sites) are difficult to distinguish by RNA sequencing unless splicing junctions are previously known.

MuSICA 2 aims at shotgun sequencing ~800 full-length cDNA clones using one flow-cell lane of Illumina GA. First, target full-length cDNA clones are amplified by PCR, and then they are mixed by equal volumes regardless of PCR efficiency. The mixture is nebulized to yield a shotgun library, which is then sequenced by Illumina GA using one flow-cell lane. Finally, MuSICA 2 assembles the shotgun reads and Sanger reads from both ends of the cDNA clones (recommended; at least either end is required to associate the output contigs with individual clones), and reconstructs their cDNA sequences with the help of the reference genome sequence. The key feature of MuSICA 2 is that it assembles shotgun reads in a hybrid way of de novo assembly and reference assembly in which shotgun reads are aligned against the reference genome. The hybrid strategy benefits both from the advantages of de novo assembly and reference assembly.

Download Source Code

Since MuSICA 2 was implemented in Perl, you do not have to compile source codes. Download the source code from the link below. Please see README.TXT in the archive for installation and usage. See below for prerequisites and manual.


Velvet and BLAT are prerequisites for MuSICA 2. You have to install and configure them before you run MuSICA 2. Velvet can be replaced with any other de novo short-read assembler, but we recommend use of Velvet for the time being because our benchmark test showed that it was the best assembler that works with MuSICA 2.



Ver 2.1.1, 14 Jul 2010: Small changes conforming with qmake standard use.
Ver 2.1.0, 6 Mar 2009: Significantly reengineered due to a drastic change in the algorithm.
Ver 1.1.0, 7 Jun 2008: Initial Release


Download Suppelmentary Data

Raw data used in the above publication can be downloaded from the link below. The same data in SRF format is also deposited in Short Read Archive. The raw data below is not required to run MuSICA2 but provided for those interested (probably tool developers). If you need other data, please contact to the authors.

  • Reginaldo Kuroshu (
  • Masahiro Kasahara (
  • Yutaka Suzuki (
  • Shin-ichi Morishita (

Old Releases

These releases are provided for archival purposes only. We do not support old releases.