======================== = SSAHA2 version 2.5.5 = ======================== by Hannes Ponstingl, Adam Spargo and Zemin Ning. Copyright (C) 2003-2011 The Wellcome Trust Sanger Institute, Cambridge, UK. All Rights Reserved. SSAHA2 is a package combining SSAHA with cross_match developed by Phil Green at the University of Washington. Reference: Ning Z, Cox AJ, Mullikin JC. SSAHA: a fast search method for large DNAdatabases. Genome Res. 2001 Oct;11(10):1725-9. DESCRIPTION: ssaha2Build This program constructs the hashtable required by the other ssaha2 programs. This provides an index for the given subject sequence. ssaha2 This program aligns query sequences against a subject hashtable. ssahaSNP ssahaSNP is a polymorphism detection tool. It detects homozygous SNPs and indels by aligning shotgun reads to the finished genome sequence. From the best alignment, SNP candidates are screened, taking into account the quality value of the bases with variation as well as the quality values in the neighbouring bases, using neighbourhood quality standard (NQS). USAGE: ************************************************************* * NOTE: SSAHA2 & SSAHASNP COMMAND LINE FORMATS HAVE CHANGED * * with version 1.0.9 * ************************************************************* ssaha2Build [OPTIONS] -save ssaha2 [OPTIONS] -save [] ssaha2 [OPTIONS] [] ssahaSNP [OPTIONS] -save ssahaSNP [OPTIONS] where and are fasta or fastq files with the reference (subject) and query sequence files. is the root name of the hash table files created from the reference sequence using ssaha2Build. The paired-end module invoked with the -pair option (see below) expects two query files with file containing the first and containing the second mate of each pair. The mates of a paired-end read must occur at the same postion in the sequence of reads in and . The paired- end module is not available for ssahaSNP. OPTIONS: -h, -help Print this page. -v, -version Print version information. -c, -cookbook Print some example parameter sets, suitable for common tasks. All other options, except the '-solexa' and '-454' flags and the '-output ssaha2 cigar' option, are key-value pairs. Options are described below (default values in brackets): -save ssaha2Build: Root name of the files to which the hash table is saved. The set of files have the extensions FILENAME.head FILENAME.body FILENAME.name FILENAME.base FILENAME.size ssaha2, ssahaSNP: Read hash table from files created by ssaha2Build. If -save is not specified then the first file name specifies the reference sequence for which a hash table is constructed on the fly -kmer Word size for ssaha hashing (13). -skip Step size for ssaha hashing (13). -ckmer Word size for cross_match matching (10). -cmatch Minimum match length for cross_match matching (14). -cut Number of repeats allowed before this kmer is ignored (10000). -seeds Number of kmer matches required to flag a hit (5). -depth Number of hits to consider for alignment (50). -memory: Memory assigned in MBs for the alignment matrix (200). -score Minimum score for match to be reported (30). -identity Minimum identity (as percentage) for match to be reported (50.000000). -port Port number for server (60000). -align If set to > 0, output graphical alignment (0). If set to 2 and -solexa flag is set: output also quality score. -edge (obsolete as of release 2.0.0) Augment hit by this many bases before alignment (200). -array: Memory assigned in bytes for frequency arrays (4000000). -start: first sequence to process in query (0). -end: last sequence to process in query, 0 means process all (0). -sense (obsolete as of release 2.0.0) Allow really patchy hits to go for alignment (0). -best If set to 1, only report the best alignment for each match, if multiple best scores report all (65). If set to a negative value '-best -' report alignment only if there are at most best mappings. -454: (see -rtype) Tune for 454 reads (0). Implies '-skip 3 -seeds 2 -score 30 -sense 1 -cmatch 10 -ckmer 6' -NQS: Use NQS to filter SNPs if set to 1, otherwise output all candidates (1). -quality: Quality value to use for variation base in NQS (23). -tags: If set to 1, prefix added to output summary lines to aid parsing, the prefix depends upon the chosen output format, e.g. if output is ssaha2 then the prefix is ALIGNMENT (1). -output: ssaha2 - original ssaha2 line only (default) sugar - Simple UnGapped Alignment Report cigar - Compact Idiosyncratic Gapped Alignment Report vulgar - Verbose Useful Labelled Gapped Alignment Report psl - Tab separated format similar to BLT http://genome.ucsc.edu/goldenPath/help/customTrack.html pslx - Tab separated format with sequence gff - http://www.sanger.ac.uk/Software/formats/GFF/ ssaha2 cigar - alternate between ssaha2 and cigar format lines aln - Tony Cox' alignment output format. sam - SAM format (http://samtools.sourceforge.net). Output is written to a file specified with the -oufile flag. sam_soft - SAM format using soft clipping. The entire read is output./n/n for a full description of output formats see: http://www.sanger.ac.uk/Software/analysis/SSAHA2/formats.shtml -name Flag that modifies option '-output cigar' such that read name and length are also reported when there was no hit found. -diff: Output all hits within diff of the best (-1). -udiff: Ignore best hit if second best score within udiff (0). -fix: If set to 1, fix -edge, -seeds, -score so that they are not updated according to read length in ssahaSNP (0). -disk: 0: map all files into memory including the query sequences. (0) 1: read hashtable body, the subject and query sequences from disk rather than loading them into memory. 2: like 1 but the subject sequences are read into memory. The body is left on the disk. In both cases the query sequence file is not mapped into memory. -weight: If >0, apply this much weighting to rare kmers (0). -solexa: (see -rtype) implies (ssaha2 and ssahaSNP only): -kmer 13 -skip 2 -seeds 2 -score 12 -cmatch 9 -ckmer 6. Top scoring hits with lower quality at the mismatch positions have their Smith-Waterman score incremented by 1. Mapping scores are changed accordingly. SsahaSNP reports in such cases only the top scoring hit (no Repeat lines). -rtype: solexa - like -solexa flag 454 - like -454 flag abi - tunes for ABI reads (default) -pair ,: Map paired-end reads with an insert length in the interval [,]. The reads for each mate are in a separate input file named by the last two tokens of the command line. The sequence for both mates are read in the same direction. Paired-end reads are reported if the mates match within bases from each other, but at least bases apart. No mate is reported, if the mapping is ambiguous, i.e. if there are multipe hits with the same Smith-Waterman alignment score in the specified distance range. If hits are found only for one mate, the highest scoring hit is reported for this mate unless it is ambiguous. -outfile Output file for output in SAM format or, if the -pair option is set, for mate pairs that map outside the specified distance range unambiguously (each with a mapping score > 30, or a value set with the -mthresh option below). -mthresh Threshold in the mapping score for mate pairs to be reported that map outside the distance range (see option -outfile). is an integer between 0 and 50 (default: 30). This option requires the -pair option to be set. -multi Flag modifying the way paired-end reads with multiple (repetitive) mappings are reported. If rand_seed == 0: skip such pairs entirely. if rand_seed> 0: select one pair at random. seeds the random number generator. ========== Change Log from version 2.0 onwards ========== Release 2.5.5 (27 September 2011) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - Limited the maximum score in SAM output to 254 (previously 255). (2.5.5: 27 September 2011, ssaha2 hp3) Release 2.5.4 (30 June 2011) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - Fixed broken clipping in the CIGAR line of the SAM output when pairs with different read lengths were mapped. The assumed read lenght was always that of the 2nd mate of the pair. - Fixed read name mix up between the mates of a pair in SAM output format when the 2nd mate was not mapped. (2.5.4: 30 June 2011, ssaha2 hp3) Release 2.5.3 (01 September 2010) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - Modified the -best option wich now now takes negative values '-best -' in which case the alignment is reported only if there are at most best mappings. (2.5.3: 01 September 2010, ssaha2 hp3) Release 2.5.2 (26 May 2010) ~~~~~~~~~~~~~~~~~~~~~~~~~~~ - Fixed bugs where program execution stopped early on some FASTQ files when the -disk 1 option was used. (2.5.2: 26 May 2010, ssaha2 hp3) Release 2.5.1 (8 February 2010) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - Bug fix concerning mapping score of paired-end reads in most output formats (with the exception of the SAM output format): The Mapping score of one of the mates of a pair (the one with fewer k-mer word hits across the genome) could be too high. Both mates of a pair are now assigned the same mapping score analogous to the SAM output format. (2.5.1: 8 February 2010, ssaha2 hp3) Release 2.5 (6 October 2009) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - added documentation to version 2.4.2 and released as version 2.5 (2.5: 6 October 2009, ssaha2 hp3) Release 2.4.2 (28 September 2009) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - Fixed a core dump with option -output SAM when query reads were given in FASTA files without quality values. - Fixed SAM output extra file '-output sam -outfile ' for single-end reads (i.e. -pair option not specified). In previous versions ignored the -outfile option if the -pair option was not specified. As a consequence mappings of single-end reads was writtend to standard output even for the SAM format. - Fixed PSLX output format. (2.4.2: 28 September 2009, ssaha2 hp3) Version 2.4.1.9 (18 September 2009) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - Re-wrote broken output of PSL format. Looks like PSL output never worked. PSLX output is still not working. (2.4.1.9: 18 September 2009, ssaha2 hp3) Version 2.4.1.8 (16 September 2009) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - Fixed -disk 1 and -disk 2 flags which were broken for paired-end reads. (2.4.1.8: 16 September 2009, ssaha2 hp3) Version 2.4.1.7 (15 September 2009) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - Fixed a bug introduced in version 2.4.1.4 that broke the SAM output of alignments on the reverse genomic strand. (2.4.1.7: 15 September 2009, ssaha2 hp3) Version 2.4.1.6 (14 September 2009) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - Fixed bug introduced in version 2.4.1.3 that broke mapping of single-end reads. (2.4.1.6: 14 September 2009, ssaha2 hp3) Version 2.4.1.5 (11 September 2009) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - changed -kmer and -skip default paramters for -rtype abi to -kmer 13 -skip 13 -solexa, -454 and -rtype options can now be specified with ssaha2Build as well as ssaha2. I.e. -kmer and -skip no loger have to be explicitly specified with ssaha2Build. This means one can build a hash table with ssaha2Build -454 -save the run the mapping with something like ssaha2 -454 -output cigar -save - fixed a bug in SAM output format that occasionally produced two tabs instead of a single tab between the sequence of base qualty values and the ssaha2 defined 'AS' field. (2.4.1.5: 11 September 2009, ssaha2 hp3) Version 2.4.1.4 (10 September 2009) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - Fixed a bug in the SAM output with soft clipping (option '-output sam_soft'). The 2nd mate was still hard clipped. (2.4.1.4: 10 September 2009, ssaha2 hp3) Version 2.4.1.3 (1 September 2009) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - "proper" pairs are now assigned the average score rather than the sum of scores of the mates. - Unmapped reads are now output in SAM format. Hitherto their output had been suppressed. - new output option '-output sam_soft' produced SAM output with 'soft clipping', i.e. the sequence of the entire read is ouptut. '-output sam' only outputs the aligned segment of the read (hard clipping). (2.4.1.3: 1 September 2009, ssaha2 hp3) Version 2.4.1.2 (28 August 2009) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - removed the constraint that paired-end reads must have identical sequence lengths. - fixed a bug in the alignment display (option -align 1) of paired-end reads. (2.4.1.2: 28 August 2009, ssaha2 hp3) Version 2.4.1.1 (27 August 2009) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - added option '-multi ' for the random selection of a read-pair in cases were there are multiple mappings with the same score. (2.4.1.1: 27 August 2009, ssaha2 hp3) Release 2.4.1 (25 August 2009) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - fixed a few things concerning the SAM output format: i) Bits 0x40 (read is first in pair) and 0x80 (read is 2nd in pair) of the binary FLAG field were set inconsistently due to an initialisation issue. ii) The read name in the QNAME files is now stripped of any '/1' and '/2' tags. - This version still requires paired-end reads to be of the same length. Where this is not the case an error message is output. Mates of differing lengths can be processed using ssaha_pileup. (2.4.1: 25 August 2009, ssaha2 hp3) Release 2.4 (21 Juli 2009) ~~~~~~~~~~~~~~~~~~~~~~~~~~ - the SAM output format now produces a new mapping score in the MAPQ-field of the SAM-format specification. - fixed a bug that resulted in core dumps when the query read files for paired-end read did not contain quality values, i.e. when the read files where in FASTA rather than FASTQ format. (2.4: 21 July 2009, ssaha2 hp3) Release 2.3.2.1 (27 April 2009) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - ssaha2 -kmer and -skip options are no longer overwritten by the parameters of the hash table when the -save option is used. Now ssaha2 exits with an error message if the kmer word lenght and skip step size specified on the command line with the -kmer and -skip options do not agree with the parameters used for the generation of the hash table. - sam output is either all to standard output or to a file specified with -output . - fixed a bug in the 'aln and 'sam' output format that occurred when query reads were in FASTA format and did not have quality values associated with them. A default quality value of 40 is output in such cases. (2.3.2.1: 27 April 2009, ssaha2 hp3) Release 2.3.2 (22 April 2009) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - fixed two bugs in SAM output format (2.3.2: 22 April 2009, ssaha2 hp3) Release 2.3.1 (6 April 2009) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - added output in SAM format (http://samtools.sourceforge.net) with the option '-output sam'. The SSAHA2 mapping score is output in an optional field with tag 'MS' (not defined by SAM-format spec). (2.3.1: 6 April 2009, ssaha2 hp3) Release 2.3.0.1 (3 April 2009) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - fixed a bug in version 2.3 that occurred for paired-end reads. Quality values following a quality value of 0 in the read were undefined. (2.3.1.0.1: 3 April 2009, ssaha2 hp3) Release 2.3 (6 March 2009) ~~~~~~~~~~~~~~~~~~~~~~~~~~~ - Added '-mthresh ' option by which a threshold in the mapping score can be set above which mate pairs are reported that map outside the insert size range. 0<= MSCORE <= 50. The default is -mthresh 30. - fixed a bug introduced in 2.2 that caused the assignment of wrong mapping scores for mate pairs. (2.3: 6 March 2009, ssaha2 hp3) Release 2.2.1 (16 February 2009) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - fixed bug introduced in version 2.2 where reads with > 5000 bases matched over the entire length in which case execution stopped with the message "position_table - Query probably contains unusual characters". (2.2.1: 16 February 2009, ssaha2 hp3) Release 2.2 (29 January 2009) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - added '-disk 2' option where hash table body and the query sequences are left on disk and the hashtable header and the reference sequences are kept in memory. - The '-disk 0' option now keeps the reference sequences in memory. - The paired-end read module (invoked with '-pair a,b') now requires separate input files for the reads of the two mates. The direction of the sequences are the same for both mates. - added Tony Cox' output format '-output aln'. - minor fixes that improved the accuracy of ssaha2. - Fix of a bug (introduced in version 2.0.0) that had ssaha2 stopping execution with the message "position_table - Query probably contains unusual characters" on some longer (>5000 bases) query reads. (2.2: 29 January 2009, ssaha2 hp3) Release 2.1 (18 September 2008) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - implemented paired-end read module with option '-pair a,b' specifying an insert range and '-outfil' specifying an output file for pairs mapping outside this range. - '-solexa' and '-rtype solexa' flags now imply '-kmer 13 -skip 2 -seeds 2 -score 12 -cmatch 9 -ckmer 6' These defaults can be overwritten, i.e. '-rtype solexa -kmer 12'. - reduced memory usage for query files with a large number of short reads. - Occasionally the first few bases are missed in the Smith-Waterman alignment if these bases contain mismatches. This version restores the full alignment if the -solexa (-rtype solexa) flag is set and the Smith-Waterman alignment starts (ends) at the 3rd or 4th (to last) base. - Fixed a bug in the PSL and PSLX output formats (wrong block starts). (2.1: 18 September 2008, ssaha2 hp3) Release 2.0.0 (16 May 2008) ~~~~~~~~~~~~~~~~~~~~~~~~~~~ - improved speed for shorter reads (-solexa and -454 flags): Changes were made to the filtering of potentially matching segments before they are passed to the Smith-Waterman alignment. Potetial matches are filtered out if the number of matching k-mer words (seeds) falls below MAXSEEDS - 5 where MAXSEEDS is the maximum in the number of seeds observed so far for the query sequence. ABI reads (default) are processed as before. - -edge option is now obsolete. Matching segments are now extended to the query read length plus 10 bases on either side. - -solexa flag now implies -cmatch 9 (previously -cmatch 10), i.e. overall '-seeds 2 -score 12 -cmatch 9 -ckmer 6'. (2.0.0: 16 May 2008, ssaha2 hp3)