Bio::Tools::Lucy - Object for analyzing the output from Lucy, a vector and quality trimming program from TIGR # Create the Lucy object from an existing Lucy output file @params = ('seqfile' => 'lucy.seq', 'lucy_verbose' => 1); $lucyObj = Bio::Tools::Lucy->new(@params); # Get names of all sequences $names = $lucyObj->get_sequence_names(); # Print seq and qual values for sequences >400 bp in order to run CAP3 foreach $name (@$names) { next unless $lucyObj->length_clear($name) > 400; print SEQ ">$name\n", $lucyObj->sequence($name), "\n"; print QUAL ">$name\n", $lucyObj->quality($name), "\n"; } # Get an array of Bio::PrimarySeq objects @seqObjs = $lucyObj->get_Seq_Objs(); Bio::Tools::Lucy.pm provides methods for analyzing the sequence and quality values generated by Lucy program from TIGR. Lucy will identify vector, poly-A/T tails, and poor quality regions in a sequence. (www.genomics.purdue.edu/gcg/other/lucy.pdf) The input to Lucy can be the Phred sequence and quality files generated from running Phred on a set of chromatograms. Lucy can be obtained (free of charge to academic users) from www.tigr.org/softlab There are a few methods that will only be available if you make some minor changes to the source for Lucy and then recompile. The changes are in the 'lucy.c' file and there is a diff between the original and the modified file in the Appendix Please contact the author of this module if you have any problems making these modifications. You do not have to make these modifications to use this module.

Creating a Lucy object
@params = ('seqfile' => 'lucy.seq', 'adv_stderr' => 1, 'fwd_desig' => '_F', 'rev_desig' => '_R'); $lucyObj = Bio::Tools::Lucy->new(@params);

Using a Lucy object
You should get an array with the sequence names in order to use accessor methods. Note: The Lucy binary program will fail unless the sequence names provided as input are unique. $names_ref = $lucyObj->get_sequence_names(); This code snippet will produce a Fasta format file with sequence lengths and %GC in the description line. foreach $name (@$names) { print FILE ">$name\t", $lucyObj->length_clear($name), "\t", $lucyObj->per_GC($name), "\n", $lucyObj->sequence($name), "\n"; } Print seq and qual values for sequences >400 bp in order to assemble them with CAP3 (or other assembler). foreach $name (@$names) { next unless $lucyObj->length_clear($name) > 400; print SEQ ">$name\n", $lucyObj->sequence($name), "\n"; print QUAL ">$name\n", $lucyObj->quality($name), "\n"; } Get all the sequences as Bio::PrimarySeq objects (eg., for use with Bio::Tools::Blast to perform BLAST). @seqObjs = $lucyObj->get_Seq_Objs(); Or use only those sequences that are full length and have a Poly-A tail. foreach $name (@$names) { next unless ($lucyObj->full_length($name) and $lucy->polyA($name)); push @seqObjs, $lucyObj->get_Seq_Obj($name); } Get the names of those sequences that were rejected by Lucy. $rejects_ref = $lucyObj->get_rejects(); Print the names of the rejects and 1 letter code for reason they were rejected. foreach $key (sort keys %$rejects_ref) { print "$key: ", $rejects_ref->{$key}; } There is a lot of other information available about the sequences analyzed by Lucy (see APPENDIX). This module can be used with the DBI module to store this sequence information in a database.

Mailing Lists
User feedback is an integral part of the evolution of this and other Bioperl modules. Send your comments and suggestions preferably to one of the Bioperl mailing lists. Your participation is much appreciated. bioperl-l@bioperl.org - General discussion http://bio.perl.org/MailList.html - About the mailing lists

Reporting Bugs
Report bugs to the Bioperl bug tracking system to help us keep track the bugs and their resolution. Bug reports can be submitted via email or the web: bioperl-bugs@bio.perl.org http://bugzilla.bioperl.org/ Andrew G. Walsh paeruginosa@hotmail.com Methods available to Lucy objects are described below. Please note that any method beginning with an underscore is considered internal and should not be called directly.

new
Title : new Usage : $lucyObj = Bio::Tools::Lucy->new(seqfile => lucy.seq, rev_desig => '_R', fwd_desig => '_F') Function: creates a Lucy object from Lucy analysis files Returns : reference to Bio::Tools::Lucy object Args : seqfile Fasta sequence file generated by Lucy qualfile Quality values file generated by Lucy infofile Info file created when Lucy is run with -debug 'infofile' option stderrfile Standard error captured from Lucy when Lucy is run with -info option and STDERR is directed to stderrfile (ie. lucy ... 2> stderrfile). Info in this file will include sequences dropped for low quality. If you've modified Lucy source (see adv_stderr below), it will also include info on which sequences were dropped because they were vector, too short, had no insert, and whether a poly-A tail was found (if Lucy was run with -cdna option). lucy_verbose verbosity level (0-1). fwd_desig The string used to determine whether sequence is a forward read. The parser will assume that this match will occus at the end of the sequence name string. rev_desig As above, for reverse reads. adv_stderr Can be set to a true value (1). Will only work if you have modified the Lucy source code as outlined in DESCRIPTION and capture the standard error from Lucy. If you don't provide filenames for qualfile, infofile or stderrfile, the module will assume that .qual, .info, and .stderr are the file extensions and search in the same directory as the .seq file for these files. For example, if you create a Lucy object with $lucyObj = Bio::Tools::Lucy->new(seqfile =>lucy.seq), the module will find lucy.qual, lucy.info and lucy.stderr. You can omit any or all of the quality, info or stderr files, but you will not be able to use all of the object methods (see method documentation below).

_parse
Title : _parse Usage : n/a (internal function) Function: called by new() to parse Lucy output files Returns : nothing Args : none

get_Seq_Objs
Title : get_Seq_Objs Usage : $lucyObj->get_Seq_Objs() Function: returns an array of references to Bio::PrimarySeq objects where -id = 'sequence name' and -seq = 'sequence' Returns : array of Bio::PrimarySeq objects Args : none

get_Seq_Obj
Title : get_Seq_Obj Usage : $lucyObj->get_Seq_Obj($seqname) Function: returns reference to a Bio::PrimarySeq object where -id = 'sequence name' and -seq = 'sequence' Returns : reference to Bio::PrimarySeq object Args : name of a sequence

get_sequence_names
Title : get_sequence_names Usage : $lucyObj->get_sequence_names Function: returns reference to an array of names of the sequences analyzed by Lucy. These names are required for most of the accessor methods. Note: The Lucy binary will fail unless sequence names are unique. Returns : array reference Args : none

sequence
Title : sequence Usage : $lucyObj->sequence($seqname) Function: returns the DNA sequence of one of the sequences analyzed by Lucy. Returns : string Args : name of a sequence

quality
Title : quality Usage : $lucyObj->quality($seqname) Function: returns the quality values of one of the sequences analyzed by Lucy. This method depends on the user having provided a quality file. Returns : string Args : name of a sequence

avg_quality
Title : avg_quality Usage : $lucyObj->avg_quality($seqname) Function: returns the average quality value for one of the sequences analyzed by Lucy. Returns : float Args : name of a sequence

direction
Title : direction Usage : $lucyObj->direction($seqname) Function: returns the direction for one of the sequences analyzed by Lucy providing that 'fwd_desig' or 'rev_desig' were set when the Lucy object was created. Strings returned are: 'F' for forward, 'R' for reverse. Returns : string Args : name of a sequence

length_raw
Title : length_raw Usage : $lucyObj->length_raw($seqname) Function: returns the length of a DNA sequence prior to quality/ vector trimming by Lucy. Returns : integer Args : name of a sequence

length_clear
Title : length_clear Usage : $lucyObj->length_clear($seqname) Function: returns the length of a DNA sequence following quality/ vector trimming by Lucy. Returns : integer Args : name of a sequence

start_clear
Title : start_clear Usage : $lucyObj->start_clear($seqname) Function: returns the beginning position of good quality, vector free DNA sequence determined by Lucy. Returns : integer Args : name of a sequence

end_clear
Title : end_clear Usage : $lucyObj->end_clear($seqname) Function: returns the ending position of good quality, vector free DNA sequence determined by Lucy. Returns : integer Args : name of a sequence

per_GC
Title : per_GC Usage : $lucyObj->per_GC($seqname) Function: returns the percente of the good quality, vector free DNA sequence determined by Lucy. Returns : float Args : name of a sequence

full_length
Title : full_length Usage : $lucyObj->full_length($seqname) Function: returns the truth value for whether or not the sequence read was full length (ie. vector present on both ends of read). This method depends on the user having provided the 'info' file (Lucy must be run with the -debug 'info_filename' option to get this file). Returns : boolean Args : name of a sequence

polyA
Title : polyA Usage : $lucyObj->polyA($seqname) Function: returns the truth value for whether or not a poly-A tail was detected and clipped by Lucy. This method depends on the user having modified the source for Lucy as outlined in DESCRIPTION and invoking Lucy with the -cdna option and saving the standard error. Note, the final sequence will not show the poly-A/T region. Returns : boolean Args : name of a sequence

get_rejects
Title : get_rejects Usage : $lucyObj->get_rejects() Function: returns a hash containing names of rejects and a 1 letter code for the reason Lucy rejected the sequence. Q- rejected because of low quality values S- sequence was short V- sequence was vector E- sequence was empty P- poly-A/T trimming caused sequence to be too short In order to get the rejects, you must provide a file with the standard error from Lucy. You will only get the quality category rejects unless you have modified the source and recompiled Lucy as outlined in DESCRIPTION. Returns : hash reference Args : none

Diff for Lucy source code
352a353,354 > /* AGW added next line */ > fprintf(stderr, "Empty: %s\n", seqs[i].name); 639a642,643 > /* AGW added next line */ > fprintf(stderr, "Short/ no insert: %s\n", seqs[i].name); 678c682,686 < if (left) seqs[i].left+=left; --- > if (left) { > seqs[i].left+=left; > /* AGW added next line */ > fprintf(stderr, "%s has PolyA (left).\n", seqs[i].name); > } 681c689,693 < if (right) seqs[i].right-=right; --- > if (right) { > seqs[i].right-=right; > /* AGW added next line */ > fprintf(stderr, "%s has PolyA (right).\n", seqs[i].name); > } 682a695,696 > /* AGW added next line */ > fprintf(stderr, "Dropped PolyA: %s\n", seqs[i].name); 734a749,750 > /* AGW added next line */ > fprintf(stderr, "Vector: %s\n", seqs[i].name);