12 my $polyAChoice = 0; # 0, default, pad polyA tails for all isoforms; 1, --no-polyA; 2, --no-polyA-subset
13 my $no_polyA = 0; # for option --no-polyA
22 GetOptions("gtf=s" => \$gtfF,
23 "transcript-to-gene-map=s" => \$mappingF,
24 "no-polyA" => \$no_polyA,
25 "no-polyA-subset=s" => \$subsetFile,
26 "polyA-length=i" => \$polyALen,
27 "bowtie-path=s" => \$bowtie_path,
28 "no-bowtie" => \$no_bowtie,
29 "no-ntog" => \$no_ntog,
31 "h|help" => \$help) or pod2usage(-exitval => 2, -verbose => 2);
33 pod2usage(-verbose => 2) if ($help == 1);
34 pod2usage(-msg => "Set --no-polyA & --no-polyA-subset at the same time!", -exitval => 2, -verbose => 2) if ($no_polyA == 1 && $subsetFile ne '');
35 pod2usage(-msg => "Invalid number of arguments!", -exitval => 2, -verbose => 2) if (scalar(@ARGV) != 2);
36 pod2usage(-msg => "If bowtie is used, --no-ntog cannot be set!", -exitval => 2, -verbose => 2) if (!$no_bowtie && $no_ntog);
38 if ($no_bowtie && $bowtie_path ne "") { print "Warning: If bowtie is not used, no need to set --bowtie-path option!\n"; }
42 if ($gtfF ne "") { $type = 0; }
45 my @list = split(/,/, $ARGV[0]);
46 my $size = scalar(@list);
48 if ($size == 1 && (-d $list[0])) {
50 @list = (<$dir/*.fa>, <$dir/*.fasta>);
51 $size = scalar(@list);
54 if ($no_polyA) { $polyAChoice = 1 }
55 elsif ($subsetFile ne "") { $polyAChoice = 2; }
57 if ($bowtie_path ne "") { $bowtie_path .= "/"; }
59 my ($fn, $dir, $suf) = fileparse($0);
64 $command = $dir."rsem-extract-reference-transcripts $ARGV[1] $quiet $gtfF";
65 if ($mappingF ne "") { $command .= " 1 $mappingF"; }
66 else { $command .= " 0"; }
69 $status = system($command);
71 print "rsem-extract-reference-transcripts failed! Please check if you provide correct parameters/options for the pipeline!\n";
78 $command = $dir."rsem-synthesis-reference-transcripts $ARGV[1] $quiet";
79 if ($mappingF ne "") { $command .= " 1 $mappingF"; }
80 else { $command .= " 0"; }
83 $status = system($command);
85 print "rsem-synthesis-reference-transcripts failed! Please check if you provide correct parameters/options for the pipeline!\n";
91 $command = $dir."rsem-preref $ARGV[1].transcripts.fa $polyAChoice $ARGV[1] -l $polyALen";
92 if ($polyAChoice == 2) { $command .= " -f $subsetFile"; }
93 if ($no_ntog) { $command .= " --no-ntog"; }
94 if ($quiet) { $command .= " -q"; }
97 $status = system($command);
99 print "rsem-preref failed! Please check if you provide correct parameters/options for the pipeline!\n";
105 $command = $bowtie_path."bowtie-build -f";
106 if ($quiet) { $command .= " -q"; }
107 $command .= " $ARGV[1].idx.fa $ARGV[1]";
110 $status = system($command);
112 print "bowtie-build failed! Please check if you provide correct parameters/options for the pipeline!\n";
122 rsem-prepare-reference
128 rsem-prepare-reference [options] reference_fasta_file(s) reference_name
136 =item B<reference_fasta_file(s)>
138 Either a comma-separated list of FASTA formatted files OR a directory name. If a directory name is specified, RSEM will read all files with suffix ".fa" or ".fasta" in this directory. The files should contain either the sequences of transcripts or an entire genome, depending on whether the --gtf option is used.
140 =item B<reference name>
142 The name of the reference used. RSEM will generate several reference-related files that are prefixed by this name. This name can contain path information (e.g. /ref/mm9).
150 =item B<--gtf> <file>
152 If this option is on, RSEM assumes that 'reference_fasta_file(s)' contains the sequence of a genome, and will extract transcript reference sequences using the gene annotations specified in <file>, which should be in GTF format.
154 If this option is off, RSEM will assume 'reference_fasta_file(s)' contains the reference transcripts. In this case, RSEM assumes that name of each sequence in the FASTA files is its transcript_id.
158 =item B<--transcript-to-gene-map> <file>
160 Use information from <file> to map from transcript (isoform) ids to gene ids.
161 Each line of <file> should be of the form:
163 gene_id transcript_id
165 with the two fields separated by a tab character.
167 If you are using a GTF file for the "UCSC Genes" gene set from the UCSC Genome Browser, then the "knownIsoforms.txt" file (obtained from the "Downloads" section of the UCSC Genome Browser site) is of this format.
169 If this option is off, then the mapping of isoforms to genes depends on whether the --gtf option is specified. If --gtf is specified, then RSEM uses the "gene_id" and "transcript_id" attributes in the GTF file. Otherwise, RSEM assumes that each sequence in the reference sequence files is a separate gene.
175 Do not add poly(A) tails to the end of reference isoforms. (Default: add poly(A) tails to all transcripts)
177 =item B<--no-polyA-subset> <file>
179 Add poly(A) tails to all transcripts except those listed in <file>. <file> is a file containing a list of transcript_ids. (Default: add poly(A) tails to all transcripts. This option cannot be used with '--no-polyA'.)
181 =item B<--polyA-length> <int>
183 The length of the poly(A) tails to be added. (Default: 125)
185 =item B<--bowtie-path> <path>
187 The path to the bowtie executables. (Default: the path to bowtie executables is assumed to be in the user's PATH environment variable)
191 Do not build Bowtie indices. Specify this option if you wish to use an alternative aligner for mapping reads to transcripts. You should align against the sequences generated in the output file 'reference_name.idx.fa'. (Default: off)
195 Disable the conversion of 'N' characters to 'G' characters in the reference sequences. This conversion is normally desired because it allows some aligners (e.g., Bowtie) to align against all positions in the reference.
200 Suppress the output of logging information. (Default: off)
204 Show help information.
210 This program extracts/preprocesses the reference sequences and builds Bowtie indices using default parameters. If an alternative aligner is to be used, then the '--no-bowtie' option should be specified to disable building Bowtie indices. This program is used in conjunction with the 'rsem-calculate-expression' program.
214 This program will generate 'reference_name.grp', 'reference_name.ti', 'reference_name.transcripts.fa', 'reference_name.seq', 'reference_name.chrlist' (if '--gtf' is on), 'reference_name.idx.fa', and corresponding Bowtie index files (unless '--no-bowtie' is specified).
216 'reference_name.grp', 'reference_name.ti', 'reference_name.seq', 'reference_name.idx.fa', and 'reference_name.chrlist' are used by RSEM internally.
218 B<'reference_name.transcripts.fa'> contains the extracted reference transcripts in FASTA format. Poly(A) tails are not added.
222 1) Suppose we have mouse RNA-Seq data and want to use the UCSC mm9 version of the mouse genome. We have downloaded the UCSC Genes transcript annotations in GTF format (as mm9.gtf) using the Table Browser and the knownIsoforms.txt file for mm9 from the UCSC Downloads. We also have all chromosome files for mm9 in the directory '/data/mm9'. We want to put the generated reference files under '/ref' with name 'mm9'. We'll add poly(A) tails with length 125. Please note that GTF files generated from UCSC's Table Browser do not contain isoform-gene relationship information. For the UCSC Genes annotation, this information can be obtained from the knownIsoforms.txt file. Suppose we want to build Bowtie indices and Bowtie executables are found in '/sw/bowtie'.
224 There are two ways to write the command:
226 rsem-prepare-reference --gtf mm9.gtf \
227 --transcript-to-gene-map knownIsoforms.txt \
228 --bowtie-path /sw/bowtie \
229 /data/mm9/chr1.fa,/data/mm9/chr2.fa,...,/data/mm9/chrM.fa \
234 rsem-prepare-reference --gtf mm9.gtf \
235 --transcript-to-gene-map knownIsoforms.txt \
236 --bowtie-path /sw/bowtie \
240 2) Suppose we only have transcripts from EST tags in 'mm9.fasta'. In addition, we also have isoform-gene information in 'mapping.txt'. We do not want to add any poly(A) tails. The reference_name will be set to 'mm9'. In addition, we do not want to build Bowtie indices, and will use an alternative aligner to align reads against the 'mm9.idx.fa' output file:
242 rsem-prepare-reference --transcript-to-gene-map mapping.txt \