15 my $polyAChoice = 0; # 0, default, pad polyA tails for all isoforms; 1, --no-polyA; 2, --no-polyA-subset
16 my $no_polyA = 0; # for option --no-polyA
23 my $bowtie2_path = "";
27 my $alleleMappingF = "";
29 GetOptions("gtf=s" => \$gtfF,
30 "transcript-to-gene-map=s" => \$mappingF,
31 "allele-to-gene-map=s" => \$alleleMappingF,
32 "no-polyA" => \$no_polyA,
33 "no-polyA-subset=s" => \$subsetFile,
34 "polyA-length=i" => \$polyALen,
35 "bowtie-path=s" => \$bowtie_path,
36 "no-bowtie" => \$no_bowtie,
37 "no-ntog" => \$no_ntog,
38 "bowtie2" => \$bowtie2,
39 "bowtie2-path=s" => \$bowtie2_path,
41 "h|help" => \$help) or pod2usage(-exitval => 2, -verbose => 2);
43 pod2usage(-verbose => 2) if ($help == 1);
44 pod2usage(-msg => "Set --no-polyA & --no-polyA-subset at the same time!", -exitval => 2, -verbose => 2) if ($no_polyA == 1 && $subsetFile ne '');
45 pod2usage(-msg => "--transcript-to-gene-map and --allele-to-gene-map are mutually exclusive!", -exitval => 2, -verbose => 2) if (($mappingF ne "") && ($alleleMappingF ne ""));
46 pod2usage(-msg => "--gtf and --allele-to-gene-map are mutually exclusive!", -exitval => 2, -verbose => 2) if (($gtfF ne "") && ($alleleMappingF ne ""));
47 pod2usage(-msg => "Invalid number of arguments!", -exitval => 2, -verbose => 2) if (scalar(@ARGV) != 2);
49 if ($bowtie2) { $no_bowtie = 1; $no_ntog = 1; }
51 pod2usage(-msg => "If bowtie is used, --no-ntog cannot be set!", -exitval => 2, -verbose => 2) if (!$no_bowtie && $no_ntog);
53 if ($no_bowtie && ($bowtie_path ne "")) { print "Warning: If bowtie is not used, no need to set --bowtie-path option!\n"; }
54 if (!$bowtie2 && ($bowtie2_path ne "")) { print "Warning: If bowtie2 is not used, no need to set --bowtie2-path option!\n"; }
58 if ($gtfF ne "") { $type = 0; }
61 my @list = split(/,/, $ARGV[0]);
62 my $size = scalar(@list);
64 if ($size == 1 && (-d $list[0])) {
66 @list = (<$dir/*.fa>, <$dir/*.fasta>);
67 $size = scalar(@list);
70 pod2usage(-msg => "reference_fasta_file(s) is empty! Please check if you provide the correct folder name or file suffixes!", -exitval => 2, -verbose => 2) if ($size <= 0);
72 if ($no_polyA) { $polyAChoice = 1 }
73 elsif ($subsetFile ne "") { $polyAChoice = 2; }
75 if ($bowtie_path ne "") { $bowtie_path .= "/"; }
76 if ($bowtie2_path ne "") { $bowtie2_path .= "/"; }
78 my $dir = "$FindBin::Bin/";
83 $command = $dir."rsem-extract-reference-transcripts $ARGV[1] $quiet $gtfF";
84 if ($mappingF ne "") { $command .= " 1 $mappingF"; }
85 else { $command .= " 0"; }
87 &runCommand($command);
91 $command = $dir."rsem-synthesis-reference-transcripts $ARGV[1] $quiet";
92 if ($mappingF ne "") { $command .= " 1 $mappingF"; }
93 elsif ($alleleMappingF ne "") { $command .= " 2 $alleleMappingF"; }
94 else { $command .= " 0"; }
96 &runCommand($command);
99 $command = $dir."rsem-preref $ARGV[1].transcripts.fa $polyAChoice $ARGV[1] -l $polyALen";
100 if ($polyAChoice == 2) { $command .= " -f $subsetFile"; }
101 if ($no_ntog) { $command .= " --no-ntog"; }
102 if ($quiet) { $command .= " -q"; }
104 &runCommand($command);
107 $command = $bowtie_path."bowtie-build -f";
108 if ($quiet) { $command .= " -q"; }
109 $command .= " $ARGV[1].idx.fa $ARGV[1]";
111 &runCommand($command);
115 $command = $bowtie2_path."bowtie2-build -f";
116 if ($quiet) { $command .= " -q"; }
117 $command .= " $ARGV[1].idx.fa $ARGV[1]";
119 &runCommand($command);
126 rsem-prepare-reference
130 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.
173 =item B<--allele-to-gene-map> <file>
175 Use information from <file> to provide gene_id and transcript_id information for each allele-specific transcript.
176 Each line of <file> should be of the form:
178 gene_id transcript_id allele_id
180 with the fields separated by a tab character.
182 This option is designed for quantifying allele-specific expression. It is only valid if '--gtf' option is not specified. allele_id should be the sequence names presented in the FASTA-formatted files.
188 Do not add poly(A) tails to the end of reference isoforms. (Default: adding poly(A) tails to all transcripts)
190 =item B<--no-polyA-subset> <file>
192 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'.)
194 =item B<--polyA-length> <int>
196 The length of the poly(A) tails to be added. (Default: 125)
198 =item B<--bowtie-path> <path>
200 The path to the Bowtie executables. (Default: the path to Bowtie executables is assumed to be in the user's PATH environment variable)
204 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)
208 Disable the conversion of 'N' characters to 'G' characters in the reference sequences prepared for aligners. This conversion is in particular desired for Bowtie aligner to align against all positions in the reference. (Default: off)
212 Build Bowtie 2 indices instead of Bowtie indices. Turn on this option will automatically turn on '--no-bowtie' and '--no-ntog' options. (Default: off)
214 =item B<--bowtie2-path>
216 The path to the Bowtie 2 executables. (Default: the path to Bowtie 2 executables is assumed to be in the user's PATH environment variable)
220 Suppress the output of logging information. (Default: off)
224 Show help information.
230 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.
234 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).
236 'reference_name.grp', 'reference_name.ti', 'reference_name.seq', and 'reference_name.chrlist' are used by RSEM internally.
238 B<'reference_name.transcripts.fa'> contains the extracted reference transcripts in FASTA format. Poly(A) tails are added unless '--no-polyA' is set.
240 B<'reference_name.idx.fa'> is used by aligners to build their own indices. If '--no-ntog' is set, this file should be identical to 'reference_name.transcripts.fa'.
244 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 'mouse_125'. 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'.
246 There are two ways to write the command:
248 rsem-prepare-reference --gtf mm9.gtf \
249 --transcript-to-gene-map knownIsoforms.txt \
250 --bowtie-path /sw/bowtie \
251 /data/mm9/chr1.fa,/data/mm9/chr2.fa,...,/data/mm9/chrM.fa \
256 rsem-prepare-reference --gtf mm9.gtf \
257 --transcript-to-gene-map knownIsoforms.txt \
258 --bowtie-path /sw/bowtie \
262 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 'mouse_0'. In addition, we do not want to build Bowtie indices, and will use an alternative aligner to align reads against the 'mouse_0.idx.fa' output file:
264 rsem-prepare-reference --transcript-to-gene-map mapping.txt \