1. What is starcode?
2. Source file list.
3. Compilation and installation.
4. Running starcode.
5. File formats.
6. License.
7. References.

Starcode is a DNA sequence clustering software. Sequence clustering is performed by finding all pairs below a Levenshtein distance metric [1]. Typically, a file containing a set of related DNA sequences is passed as input, jointly with a parameter specifying the desired cluster distance. Starcode aligns and computes the distance between all the sequence pairs [2] and prints a line for each cluster containing: canonical DNA sequence, sequence count and the list of sequences that belong to the cluster.

Starcode has many applications in the field of biology, such as DNA/RNA motif recovery, barcode clustering, sequencing error recovery, etc.

• main-starcode.c Starcode main file (parameter parsing).
• starcode.c Main starcode algorithm.
• starcode.h Main starcode algorithm public header file.
• starcode-private.h Main starcode algorithm private header file.
• trie.c Trie search and construction functions.
• trie.h Trie public header file.
• trie-private.h Trie private header file.
• Makefile Make instruction file.

To install starcode you first need to clone or manually download the repository content from github:

git clone git://github.com/gui11aume/starcode.git

the files should be downloaded in a folder named 'starcode'. To compile just change the directory to 'starcode' and run make (Mac users require 'xcode', available at the Mac Appstore):

cd starcode

make

a binary file 'starcode' will be created. You can optionally make a symbolic link to execute starcode from any directory:

sudo ln -s ./starcode /usr/bin/starcode

Starcode runs on Linux and Mac. It has not been tested on Windows.

List of arguments:

starcode [options] {[-i] INPUT_FILE | -1 PAIRED_END_FILE1 -2 PAIRED_END_FILE2} [-o OUTPUT_FILE]

-d or --distance distance

 Defines the maximum Levenshtein distance for clustering.
 When not set it is automatically computed as:
 min(8, 2 + [median seq length]/30)

-t or --threads threads

 Defines the maximum number of parallel threads.
 Default is 1.

-s or --spheres

 When specified, sphere clustering algorithm is performed in the
 clustering phase, instead of the default message passing algorithm.

-r or --cluster-ratio ratio

 Specifies the minimum sequence count ratio to cluster two matching
 sequences, i.e. the matching sequences A and B will only be
 clustered together if count(A) > ratio * count(B), assuming that
 count(A) > count(B).
 Note that this option only applies to message passing algorithm and
 ratio must be set to 1 to cluster unique input sequences together.
 Default is 5.

-q or --quiet

 Non verbose. By default, starcode prints verbose information to
 the standard error channel.

-h or --help

 Prints usage information.

-o or --output file

 Specifies output file. When not set, standard output is used instead.

--non-redundant

 Removes redundant sequences from the output. Only the canonical sequence
 of each cluster is returned.

--print-clusters

 Adds a third column to the starcode output, containing the sequences
 associated with each cluster. By default, the output contains only
 the centroid and the counts.

--seq-id

 Shows the clustered sequence numbers (1-based) following the original
 input order.

Single-file mode:

-i or --input file

 Specifies input file.

Paired-end fastq files:

-1 file1 -2 file2

 Specifies two paired-end FASTQ files for paired-end clustering mode.

Standard input is used when neither -i nor -1/-2 are set.

Consists of a file containing one sequence per line. Only the standard DNA-base characters are supported ('A', 'C', 'G', 'T'). The sequences may not contain empty spaces at the beginning or the end of the string, as these will be counted as alignment characters. The file may not contain empty lines as these will be considered as zero-length sequences. The sequences do not need to be sorted and may be repeated.

Example:

TTACTATCGATCATCATCGACTGACTACG
ACTGCATCGACTAGCTACGACTACGCTACCATCAG
TTACTATCGATCATCATCGACTGACTAGC
ACTACGACTACGACTCAGCTCACTATCAGC
GCATCGACCGCTACTACGCATACTACGACATC

If the count of the sequences is known, it may be specified in the input file using the following format:

[SEQUENCE]\t[COUNT]\n

Where '\t' denotes the TAB character and '\n' the NEWLINE character. The sequences do not need to be sorted and may be repeated as well. If a repeated sequence is found, their counts will be addded together. As before, the sequences may not contain any additional characters and the file may not contain empty lines.

Example:

TATCGACTCTATCTATCGCTGATGCGTAC       200
CGAGCCGCCGGCACGTCACGACGCATCAA       1
TAGCACCTACGCATCTCGACTATCACG         234
CGAGCCGCCGGCACGTCACGACGCATCAA       17
TGACTCTATCAGCTAC                    39

Starcode supports FASTA and FASTQ files as well. Note, however, that starcode does not use the quality factors and the only relevant information is the sequence itself. The FASTA/FASTQ labels will not be used to identify the sequences in the output file. The sequences do not need to be sorted and may be repeated.

Example FASTA:

> FASTA sequence 1 label
ATGCATCGATCACTCATCAGCTACAG
> FASTA sequence 2 label
TATCGACTATCTACGACTACATCA
> FASTA sequence 3 label
ATCATCACTCTAGCAGCGTACTCGCA
> FASTA sequence 4 label
ATGCATCGATTACTCATCAGCTACAG

Example FASTQ:

@ FASTQ sequence 1 label
CATCGAGCAGCTATGCAGCTACGAGT
+
-$#'%-#.&)%#)"".)--'*()$)%
@ FASTQ sequence 2 label
TACTGCTGATATTCAGCTCACACC
+
,*#%+#&*$-#,''+*)'&.,).,

Starcode prints a line for each detected cluster with the following format:

[CANONICAL SEQUENCE]\t[CLUSTER SIZE]\t[CLUSTER SEQUENCES]\n

Where '\t' denotes the TAB character and '\n' the NEWLINE character. 'CANONICAL SEQUENCE' is the sequence of the cluster that has more counts, 'CLUSTER SIZE' is the aggregated count of all the sequences that form the cluster, and 'CLUSTER SEQUENCES' is a list of all the cluster sequences separated by commas and in arbitrary order. The lines are printed sorted by 'CLUSTER SIZE' in descending order.

For instance, an execution with the following input and clustering distance of 3 (-d3):

TAGCTAGACGTA   250
TAGCTAGCCGTA   10
TAAGCTAGGGGT   16
ACGCGAGCGGAA   155
ACTTTAGCGGAA   1

would produce the following output:

TAGCTAGACGTA    276       TAGCTAGACGTA,TAGCTAGCCGTA,TAAGCTAGGGGT
ACGCGAGCGGAA    156       ACGCGAGCGGAA,ACTTTAGCGGAA

The same example executed with a more restrictive distance -d2 would produce the following output:

TAGCTAGACGTA    260       TAGCTAGACGTA,TAGCTAGCCGTA
ACGCGAGCGGAA    155       ACGCGAGCGGAA
TAAGCTAGGGGT    16        TAAGCTAGGGGT
ACTTTAGCGGAA    1         ACTTTAGCGGAA

In non-redundant output mode, starcode only prints the canonical sequence of each cluster, one per line. Following the example from the previous section, the output with distance 3 (-d3) would be:

  TAGCTAGACGTA
  ACGCGAGCGGAA

whereas for -d2:

  TAGCTAGACGTA
  ACGCGAGCGGAA
  TAAGCTAGGGGT
  ACTTTAGCGGAA

Starcode is licensed under the GNU General Public License, version 3 (GPLv3), for more information read the LICENSE file or refer to:

http://www.gnu.org/licenses/

[1] Levenshtein, V. (1966), 'Binary Codes Capable of Correcting Deletions, Insertions and Reversals', Soviet Physics Doklady 10, 707.

[2] Needleman, S.B. and Wunsch, C.D. (1970), 'A general method applicable to the search for similarities in the amino acid sequence of two proteins' J. Mol. Biol., 48 (3), 443-53.

If you use our software, please cite:

Zorita E, Cusco P, Filion GJ. 2015. Starcode: sequence clustering based on all-pairs search. Bioinformatics 31 (12): 1913-1919.