It can optionally build Bowtie indices (with '--bowtie' option) and/or Bowtie 2 indices (with '--bowtie2' option) using their default parameters. allele_id should be the sequence names presented in the Multi-FASTA-formatted files. The name of the reference used. Assuming STAR executable is '/sw/STAR', the command will be: STAR genome index files will be saved under '/ref/'. Please note that GTF files generated from UCSC's Table Browser do not contain isoform-gene relationship information. The SAM/BAM file declares more reference sequences (572504) than RSEM knows (88647). rsem-prepare-reference --gtf annotations.gtf --star genome.fa ref_name. It is used for selecting a training set of isoforms for prior-learning. It can also optionally build STAR indices (with '--star' option) using parameters from ENCODE3's STAR-RSEM pipeline. I have the same problem when I run RSEM without -gtf option. It is only used for STAR to build splice junctions database and not needed for Bowtie or Bowtie2. In this scenario, both STAR and Bowtie are required to build genomic indices - STAR for RNA-seq reads and Bowtie for ChIP-seq reads. This program extracts/preprocesses the reference sequences for RSEM. If you are using RSEM v1.2.24, yes, it is because we attached gene/transcript name at the end of gene/transcript id but STAR does not. RSEM will generate several reference-related files that are prefixed by this name. The reference_name is set as 'mouse_125'. RSEM/rsem-prepare-reference Go to file Cannot retrieve contributors at this time executable file 508 lines (359 sloc) 22 KB Raw Blame #!/usr/bin/env perl use Getopt::Long qw (:config no_auto_abbrev); use Pod::Usage; use File::Basename; use FindBin; use lib $FindBin::RealBin; use rsem_perl_utils; use Env qw (@PATH); (Default: 1), Suppress the output of logging information. 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', 'reference_name.n2g.idx.fa', optional Bowtie/Bowtie 2 index files, and optional STAR index files. This goes against the policy of the --gtf flag though where RSEM will assume the reference file is transcriptome.fa. Currently, Bowtie2 is not supported for prior-enhanced RSEM. I've been using STAR for awhile now so I may be able to offer some input. reference_fasta_file(s) Either a comma-separated list of Multi-FASTA formatted files OR a directory name. 1) Suppose we have mouse RNA-Seq data and want to use the UCSC mm9 version of the mouse genome. It can also optionally build STAR indices (with '--star' option) using parameters from ENCODE3's STAR-RSEM pipeline. The files should contain either the sequences of transcripts or an entire genome, depending on whether the --gtf option is used. In addition, poly(A) tails are added if '--polyA' option is set. Do not add poly(A) tails to those transcripts listed in . According to STAR's manual, its ideal value is max(ReadLength)-1, e.g. So, I manually made a gtf file, which treat each seq as an entire exon. It will be passed as the --sjdbOverhang option to STAR. RSEMindex BowtieBowtie2STARHisat2Hisat2 (Default: "mRNA"). (Default: off), The path to the HISAT2 executables. (Default: do not add poly(A) tail to any of the isoforms), The length of the poly(A) tails to be added. This program is used in conjunction with the 'rsem-calculate-expression' program. 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. 5) Suppose we only have transcripts from EST tags stored in 'mm9.fasta' and isoform-gene information stored in 'mapping.txt'. RSEM uses 'reference_name.idx.fa' to build Bowtie 2 indices and 'reference_name.n2g.idx.fa' to build Bowtie indices. RSEM will generate several reference-related files that are prefixed by this name. 1) Suppose we have mouse RNA-Seq data and want to use the UCSC mm9 version of the mouse genome. Only transcripts that match the will be extracted. (Default: 125), Only meaningful if '--polyA' is specified. The command will be: --mappability-bigwig-file /data/mm9.bigWig \, Both STAR and Bowtie's index files will be saved under '/ref/'. (Default: the path to STAR executable is assumed to be in user's PATH environment variable), Length of the genomic sequence around annotated junction. A path to Bowtie executables and a mappability file in bigWig format are required when this option is on. (Default: off), Full path to a whole-genome mappability file in bigWig format. 1) Suppose we have mouse RNA-Seq data and want to use the UCSC mm9 version of the mouse genome. It can optionally build Bowtie indices (with '--bowtie' option) and/or Bowtie 2 indices (with '--bowtie2' option) using their default parameters. In most cases, the default value of 100 will work as well as the ideal value. If '--gtf' is specified, then RSEM uses the "gene_id" and "transcript_id" attributes in the GTF file. For now, you can. Each line of should be of the form: with the two fields separated by a tab character. Selected isoforms for training set are listed in the file 'reference_name_prsem.training_tr_crd'. STAR aligner users may not want to use this option. Add poly(A) tails to the end of all reference isoforms. If a directory name is specified, RSEM will read all files with suffix ".fa" or ".fasta" in this directory. It can also optionally build STAR indices (with '--star' option) using parameters from ENCODE3's STAR-RSEM pipeline. 'reference_name.grp', 'reference_name.ti', 'reference_name.seq', and 'reference_name.chrlist' are used by RSEM internally. --transcript-to-gene-map knownIsoforms.txt \, /data/mm9/chr1.fa,/data/mm9/chr2.fa,,/data/mm9/chrM.fa \. We also have all chromosome files for mm9 in the directory '/data/mm9'. 'reference_name.grp', 'reference_name.ti', 'reference_name.seq', and 'reference_name.chrlist' are used by RSEM internally. If --gtf is specified, then RSEM uses the "gene_id" and "transcript_id" attributes in the GTF file. We want to put the generated reference files under '/ref' with name 'mouse_0'. You signed in with another tab or window. Suppose we want to build Bowtie indices and Bowtie executables are found in '/sw/bowtie'. (Default: do not add poly(A) tail to any of the isoforms), The length of the poly(A) tails to be added. (Default: 100), Build HISAT2 indices on the transcriptome according to Human Cell Atlas (HCA) SMART-Seq2 pipeline. Please note that GTF files generated from UCSC's Table Browser do not contain isoform-gene relationship information. "ENSEMBL,HAVANA". It can optionally build Bowtie indices (with '--bowtie' option) and/or Bowtie 2 indices (with '--bowtie2' option) using their default parameters. Either a comma-separated list of Multi-FASTA formatted files OR a directory name. is a file containing a list of transcript_ids. Bowtie files will have name prefix 'mouse_0_prsem'. Currently, Bowtie2 is not supported for prior-enhanced RSEM. If a directory name is specified, RSEM will read all files with suffix ".fa" or ".fasta" in this directory. The name of the reference used. to your account. rsem-prepare-reference [options] reference_fasta_file(s) reference_name ARGUMENTS reference_fasta_file(s) Either a comma-separated list of Multi-FASTA formatted files OR a directory name. Assuming their executables are under '/sw/STAR' and '/sw/Bowtie', respectively. 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 , which should be in GTF format. rsem-prepare-reference --star genome.fa ref_name. STAR aligner users may not want to use this option. (Default: off), is a comma-separated list of transcript categories, e.g. (Default: off). For visualizing the transcript-coordinate-based BAM files generated by RSEM in IGV, 'reference_name.idx.fa' should be imported as a "genome" (see Visualization section in README.md for details). (Default: off), Full path to a whole-genome mappability file in bigWig format. rsempreparereference Prepare transcript references for RSEM and optionally build BOWTIE/BOWTIE2/STAR indices. for 2x101 paired-end reads, the ideal value is 101-1=100. + . The text was updated successfully, but these errors were encountered: For the record, I ran in to this same issue. This option is designed for untypical organisms, such as viruses, whose GFF3 files only contain genes. The name of the reference used. Are you sure you want to create this branch? However, when running rsem-calculate-expression, after STAR alignment, there is an error that the BAM file contains many more sequences than RSEM knows. This conversion is in particular desired for aligners (e.g. is a comma-separated list of trusted sources, e.g. This conversion is in particular desired for aligners (e.g. It can also optionally build STAR indices (with '--star' option) using parameters from ENCODE3's STAR-RSEM pipeline. This name can contain path information (e.g. If this option is off, all sources are accepted. 'reference_name.idx.fa' and 'reference_name.n2g.idx.fa' are used by aligners to build their own indices. SYNOPSIS rsempreparereference [options] reference_fasta_file(s) reference_name ARGUMENTS . rsem-prepare-reference - Prepare transcript references for RSEM and optionally build BOWTIE/BOWTIE2/STAR/HISAT2(transcriptome) indices. Since I want to compare the results from the two transcriptomes, I don't want the result differences derived from the difference between STAR and bowtie. If a directory name is specified, RSEM will read all files with suffix ".fa" or ".fasta" in this directory. We want to add 125bp long poly(A) tails to all transcripts. Use rsem-prepare-reference to create index files for STAR. The RSEM package provides an user-friendly interface, supports threads for parallel computation of the EM algorithm, single-end and paired-end read data, quality scores, variable-length reads and RSPD estimation. It is only used for STAT to build splice junctions database and not needed for Bowtie or Bowtie2. In both cases, STAR takes the genome as input. This commit does not belong to any branch on this repository, and may belong to a fork outside of the repository. It looks like the issue is that the --trusted-sources information is not fed into STAR when it creates it's reference, and so it uses many more transcripts than RSEM is keeping in its own files. Only transcripts coming from these sources will be extracted. The user must have run 'rsem-prepare-reference' with this reference_name before running this program. This option is designed for quantifying allele-specific expression. rsem-prepare-reference - Prepare transcript references for RSEM and optionally build BOWTIE/BOWTIE2/STAR/HISAT2 (transcriptome) indices. Many Git commands accept both tag and branch names, so creating this branch may cause unexpected behavior. Poly(A) tails are not added and it may contain lower case bases in its sequences if the corresponding genomic regions are soft-masked. Each line of should be of the form: with the fields separated by a tab character. I am working under anaconda3 so i have installed RSEM using : conda install -c bioconda rsem When I run it rsem-prepare-reference --gtf Mus_musculus.GRCm38.83.gtf --bowtie Mus_musculus.GRCm38.dna.primary_assembly.fa ref/Mouse_ensembl I g. For prior-enhanced RSEM, it can build Bowtie genomic indices and select training set isoforms (with options '--prep-pRSEM' and '--mappability-bigwig-file '). Selected isoforms for training set are listed in the file 'reference_name_prsem.training_tr_crd'. For the UCSC Genes annotation, this information can be obtained from the knownIsoforms.txt file. This program extracts/preprocesses the reference sequences for RSEM and prior-enhanced RSEM. 'reference_name.transcripts.fa' contains the extracted reference transcripts in Multi-FASTA format. It can also optionally build STAR indices (with '--star' option) using parameters from ENCODE3's STAR-RSEM . sample_name The name of the sample analyzed. 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. According to STAR's manual, its ideal value is max(ReadLength)-1, e.g. This program is used in conjunction with the 'rsem-calculate-expression' program. 4) Suppose we want to prepare references for prior-enhanced RSEM in the above example. 2) Pass in annotations to STAR. RSEM will first convert it to GTF format with the file name 'reference_name.gtf'. with the fields separated by a tab character. This won't work because RSEM will expect the reference file to be transcriptome.fa even though STAR should be called with genome.fa and annotations.gtf as input. rsem-prepare-reference [options] reference_fasta_file(s) reference_name. Learn more about bidirectional Unicode characters. In this case, RSEM assumes that name of each sequence in the Multi-FASTA files is its transcript_id. Seeing that this issue has been open since 2017, it would be nice to at least have a note about this problem in the vignette, please! 'reference_name.idx.fa' and 'reference_name.n2g.idx.fa', If the whole genome is indexed for prior-enhanced RSEM, all the index files will be generated with prefix as 'reference_name_prsem'. This file is required for running prior-enhanced RSEM. Assuming STAR executable is '/sw/STAR', the command will be: STAR genome index files will be saved under '/ref/'. In this case, RSEM assumes that name of each sequence in the Multi-FASTA files is its transcript_id. 'reference_name.idx.fa' and 'reference_name.n2g.idx.fa' are used by aligners to build their own indices. The length of poly(A) tail added is specified by '--polyA-length' option. If this option is off, then the mapping of isoforms to genes depends on whether the --gtf option is specified. If a directory name is specified, RSEM will read all files with suffix ".fa" or ".fasta" in this directory. 4) Suppose we want to prepare references for prior-enhanced RSEM in the above example. Also, assuming the mappability file for mouse genome is '/data/mm9.bigWig'. allele_id should be the sequence names presented in the Multi-FASTA-formatted files. reference_fasta_file(s) Either a comma-separated list of Multi-FASTA formatted files OR a directory name. is a file containing a list of transcript_ids. However, I am worrying if the faked 'gtf' has negative effects on results. is a comma-separated list of trusted sources, e.g. (Default: off), A Boolean indicating whether to prepare reference files for pRSEM, including building Bowtie indices for a genome and selecting training set isoforms. 2) Suppose we also want to build Bowtie 2 indices in the above example and Bowtie 2 executables are found in '/sw/bowtie2', the command will be: 3) Suppose we want to build STAR indices in the above example and save index files under '/ref' with name 'mouse_0'. You signed in with another tab or window. Use information from to map from transcript (isoform) ids to gene ids. 2) Suppose we also want to build Bowtie 2 indices in the above example and Bowtie 2 executables are found in '/sw/bowtie2', the command will be: 3) Suppose we want to build STAR indices in the above example and save index files under '/ref' with name 'mouse_0'. We do not add any poly(A) tails. The files should contain either the sequences of transcripts or an entire genome, depending on whether the '--gtf' option is used. In most cases, the default value of 100 will work as well as the ideal value. RSEM is a software package for estimating gene and isoform expression levels from RNA-Seq data. /ref/mm9). Each line of should be of the form: with the two fields separated by a tab character. Use information from to map from transcript (isoform) ids to gene ids. Do not add poly(A) tails to those transcripts listed in . Already on GitHub? 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 , which should be in GTF format. 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. (Default: the path to Bowtie 2 executables is assumed to be in the user's PATH environment variable), The path to STAR's executable. (Default: the path to Bowtie executables is assumed to be in the user's PATH environment variable), The path to the Bowtie 2 executables. This program extracts/preprocesses the reference sequences for RSEM and prior-enhanced RSEM. Also, assuming the mappability file for mouse genome is '/data/mm9.bigWig'. In this case, RSEM assumes that name of each sequence in the Multi-FASTA files is its transcript_id. Each line of should be of the form: with the fields separated by a tab character. Did you find any solution? We want to put the generated reference files under '/ref' with name 'mouse_0'. It can optionally build Bowtie indices (with '--bowtie' option) and/or Bowtie 2 indices (with '--bowtie2' option) using their default parameters. privacy statement. 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 , which should be in GTF format. A tag already exists with the provided branch name. Use information from to provide gene_id and transcript_id information for each allele-specific transcript. Otherwise, RSEM assumes that each sequence in the reference sequence files is a separate gene. The index files will be used for aligning ChIP-seq reads in prior-enhanced RSEM and the training set isoforms will be used for learning prior. (Default: ""). We will fix this bug as soon as we can. If a directory name is . Have a question about this project? Well occasionally send you account related emails. If this option is off, RSEM will assume 'reference_fasta_file(s)' contains the reference transcripts. is a file containing a list of transcript_ids. Otherwise, 'reference_name.idx.fa' should be used to build the aligner's index files. RSEM will assume each gene as a unique transcript when it converts the GFF3 file into GTF format. This won't work because RSEM will expect the reference file to be transcriptome.fa even though STAR should be called with genome.fa and annotations.gtf as input. For visualizing the transcript-coordinate-based BAM files generated by RSEM in IGV, 'reference_name.idx.fa' should be imported as a "genome" (see Visualization section in README.md for details). (Default: the path to Bowtie 2 executables is assumed to be in the user's PATH environment variable), The path to STAR's executable. (Default: off), The path to the Bowtie executables. I think this is conceptually* similar to: #143 (*a mismatch between annotation gtf/gff and the genome fasta seems to cause the error, but this mismatch can happen due to various reasons), rsem-prepare-reference with STAR using Trusted Sources. Bowtie) that do not allow reads to overlap with 'N' characters in the reference sequences. In my opinion, this opens up a couple options for RSEM users that want to use STAR. It can also optionally build STAR indices (with '--star' option) using parameters from ENCODE3's STAR-RSEM pipeline. "mRNA,rRNA". This file can be either downloaded from UCSC Genome Browser or generated by GEM (Derrien et al., 2012, PLoS One). Please make sure that 'reference_name.gtf' does not exist. This program extracts/preprocesses the reference sequences for RSEM and prior-enhanced RSEM. (Default: 125), Only meaningful if '--polyA' is specified. i was under the impression that I couldn't just feed in transcriptome coordinate bam files generated with star . Assuming their executables are under '/sw/STAR' and '/sw/Bowtie', respectively. Many Git commands accept both tag and branch names, so creating this branch may cause unexpected behavior. Based on the RSEM group discussions, I ran my STAR with the following options: Code: --alignIntronMax 1 --alignIntronMin 2 --scoreDelOpen -10000 --scoreInsOpen -10000 --alignEndsType EndToEnd This becasue RSEM requires input that has been mapped to the transcriptome instead of the geneome. Suppose we want to build Bowtie indices and Bowtie executables are found in '/sw/bowtie'. Either a comma-separated list of Multi-FASTA formatted files OR a directory name. This program is used in conjunction with the 'rsem-calculate-expression' program. gene_id "TRINITY_DN22396_c2_g1_i15";transcript_id "TRINITY_DN22396_c2_g1_i15";gene_name "TRINITY_DN22396_c2_g1_i15"; http://groups.google.com/group/rsem-users. Suppose we want to build Bowtie indices and Bowtie executables are found in '/sw/bowtie'. By clicking Sign up for GitHub, you agree to our terms of service and This file is required for running prior-enhanced RSEM. A tag already exists with the provided branch name. (Default: do not add poly(A) tail to any of the isoforms), The length of the poly(A) tails to be added. I think I'll just try a workaround where I pre-filter my GTF file, but I just thought I'd bring this to your attention so you could either fix or have the tool raise an error message when -star and --trusted-sources are used together. 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. The reference_name is set as 'mouse_125'. Otherwise, 'reference_name.idx.fa' should be used to build the aligner's index files. This program extracts/preprocesses the reference sequences for RSEM. (Default: the path to HISAT2 executables is assumed to be in the user's PATH environment variable), Number of threads to use for building STAR's genome indices. If this option is off, then the mapping of isoforms to genes depends on whether the '--gtf' option is specified. The files should contain either the sequences of transcripts or an entire genome, depending on whether the '--gtf' option is used. If this option is off, then the mapping of isoforms to genes depends on whether the '--gtf' option is specified. We want to put the generated reference files under '/ref' with name 'mouse_0'. All output files are prefixed by this name (e.g., . I ran rsem-prepare-reference in '-star' mode but also using the '--trusted-sources' to limit the source to "BestRefSeq" and "Curated . For the UCSC Genes annotation, this information can be obtained from the knownIsoforms.txt file. In addition, we do not want to build Bowtie/Bowtie 2 indices, and will use an alternative aligner to align reads against either 'mouse_125.idx.fa' or 'mouse_125.idx.n2g.fa'. Use information from to map from transcript (isoform) ids to gene ids. For prior-enhanced RSEM, it can build Bowtie genomic indices and select training set isoforms (with options '--prep-pRSEM' and '--mappability-bigwig-file '). (Default: off), is a comma-separated list of transcript categories, e.g. To review, open the file in an editor that reveals hidden Unicode characters. If an alternative aligner is to be used, indices for that particular aligner can be built from either 'reference_name.idx.fa' or 'reference_name.n2g.idx.fa' (see OUTPUT for details). If an alternative aligner is to be used, indices for that particular aligner can be built from either 'reference_name.idx.fa' or 'reference_name.n2g.idx.fa' (see OUTPUT for details). We also have all chromosome files for mm9 in the directory '/data/mm9'. rsem-prepare-reference [options] reference_fasta_file(s) reference_name. 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', 'reference_name.n2g.idx.fa', optional Bowtie/Bowtie 2 index files, and optional STAR index files. In this scenario, both STAR and Bowtie are required to build genomic indices - STAR for RNA-seq reads and Bowtie for ChIP-seq reads. The annotation file is in GFF3 format instead of GTF format. Only transcripts that match the will be extracted. The length of poly(A) tail added is specified by '--polyA-length' option. RSEM will generate several reference-related files that are prefixed by this name. (Default: the path to STAR executable is assumed to be in user's PATH environment varaible), Length of the genomic sequence around annotated junction. STAR aligner users may not want to use this option. If '--gtf' is specified, then RSEM uses the "gene_id" and "transcript_id" attributes in the GTF file. Either a comma-separated list of Multi-FASTA formatted files OR a directory name. If this and '--gff3' options are off, RSEM will assume 'reference_fasta_file(s)' contains the reference transcripts. Add poly(A) tails to the end of all reference isoforms. This option is designed for untypical organisms, such as viruses, whose GFF3 files only contain genes. The annotation file is in GFF3 format instead of GTF format. Sign in gene_id "TRINITY_DN27712_c2_g1_i5";transcript_id "TRINITY_DN27712_c2_g1_i5";gene_name "TRINITY_DN27712_c2_g1_i5"; TRINITY_DN22396_c2_g1_i15 Trinity_gene exon 1 6564 . (Default: 1), Suppress the output of logging information. STAR is capable of discovering the annotations by itself. Then I can run RSEM using STAR with the 'faked' gtf. This file can be either downloaded from UCSC Genome Browser or generated by GEM (Derrien et al., 2012, PLoS One). SYNOPSIS rsem-prepare-reference [options] reference_fasta_file (s) reference_name ARGUMENTS reference_fasta_file (s) Either a comma-separated list of Multi-FASTA formatted files OR a directory name. You do not have permission to delete messages in this group, Either email addresses are anonymous for this group or you need the view member email addresses permission to view the original message, I am trying to build RSEM STAR index for ERCC92 Spike-In reference. 'reference_name.transcripts.fa' contains the extracted reference transcripts in Multi-FASTA format. I have been trying to use the --star option as well. (Default: "mRNA"). In these two files, all sequence bases are converted into upper case. '/ref/mm9'). (Default: 1), Suppress the output of logging information. 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. rsem-prepare-reference --gtf annotations.gtf --star genome.fa ref_name. If a directory name is . In these two files, all sequence bases are converted into upper case. RSEM will assume each gene as a unique transcript when it converts the GFF3 file into GTF format. In most cases, the default value of 100 will work as well as the ideal value. Sometimes, for example, when you work with spike-ins you dont have reference genome, but transcriptome instead, so maybe it is possible to run STAR from RSEM and pass parameters to it without "--sjdbGTFfile"? This option is designed for quantifying allele-specific expression. For example : rsem-prepare-reference --gtf mm9.gtf --star --star-path /sw/STAR -p 8 /data/mm9 (genome file path). (Default: off). for 2x101 paired-end reads, the ideal value is 101-1=100. To save computational time and memory resources, STAR's Output BAM file is unsorted . (Default: off), The path to the HISAT2 executables. (Default: 100), Build HISAT2 indices on the transcriptome according to Human Cell Atlas (HCA) SMART-Seq2 pipeline. rule prepare_reference: input: # reference fasta with either the entire genome or transcript sequences reference_genome="genome.fasta", output: # one of the index files created and used by rsem (required) seq="index/reference.seq", # rsem produces a number of other files which may optionally be specified as output; these may be provided so that The index files will be used for aligning ChIP-seq reads in prior-enhanced RSEM and the training set isoforms will be used for learning prior. rsempreparereference Prepare transcript references for RSEM and optionally build BOWTIE/BOWTIE2/STAR indices. allele_id should be the sequence names presented in the Multi-FASTA-formatted files. + . (Default: off), A Boolean indicating whether to prepare reference files for pRSEM, including building Bowtie indices for a genome and selecting training set isoforms. Sign up for a free GitHub account to open an issue and contact its maintainers and the community. Assuming STAR executable is '/sw/STAR', the command will be: STAR genome index files will be saved under '/ref/'. (Default: off), The path to the Bowtie executables. Only transcripts coming from these sources will be extracted. (Default: the path to Bowtie 2 executables is assumed to be in the user's PATH environment variable), The path to STAR's executable. TRINITY_DN27712_c2_g1_i5 Trinity_gene exon 1 6567 . In addition, poly(A) tails are added if '--polyA' option is set. (Default: the path to Bowtie executables is assumed to be in the user's PATH environment variable), The path to the Bowtie 2 executables. Please make sure that 'reference_name.gtf' does not exist. If this and '--gff3' options are off, RSEM will assume 'reference_fasta_file(s)' contains the reference transcripts. (Default: the path to Bowtie executables is assumed to be in the user's PATH environment variable), The path to the Bowtie 2 executables. We also have all chromosome files for mm9 in the directory '/data/mm9'. Use information from to provide gene_id and transcript_id information for each allele-specific transcript. Although it is recommended to run STAR with annotations, A. This name can contain path information (e.g. 'reference_name.grp', 'reference_name.ti', 'reference_name.seq', and 'reference_name.chrlist' are used by RSEM internally. This name can contain path information (e.g. The length of poly(A) tail added is specified by '--polyA-length' option. A path to Bowtie executables and a mappability file in bigWig format are required when this option is on. Each line of should be of the form: with the two fields separated by a tab character. Unfortunately, at least from the rsem-prepare-reference help, there doesn't seem to be any way to add this to the rsem-prepare-reference pipeline. It will be passed as the --sjdbOverhang option to STAR. is this correct and if so, besides "why?", what would be the recommended work-around? 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', 'reference_name.n2g.idx.fa', optional Bowtie/Bowtie 2 index files, and optional STAR index files. RSEM uses 'reference_name.idx.fa' to build Bowtie 2 indices and 'reference_name.n2g.idx.fa' to build Bowtie indices. RSEM will first convert it to GTF format with the file name 'reference_name.gtf'. (Default: 100), Number of threads to use for building STAR's genome indices. Bowtie files will have name prefix 'mouse_0_prsem'. It can optionally build Bowtie indices (with '--bowtie' option) and/or Bowtie 2 indices (with '--bowtie2' option) using their default parameters. for 2x101 paired-end reads, the ideal value is 101-1=100. (Default: ""). If the whole genome is indexed for prior-enhanced RSEM, all the index files will be generated with prefix as 'reference_name_prsem'. It can optionally build Bowtie indices (with '--bowtie' option) and/or Bowtie 2 indices (with '--bowtie2' option) using their default parameters. (Default: off), The path to the Bowtie executables. We want to add 125bp long poly(A) tails to all transcripts. 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. Poly(A) tails are not added and it may contain lower case bases in its sequences if the corresponding genomic regions are soft-masked. It is only valid if '--gtf' option is not specified. "mRNA,rRNA". Do not add poly(A) tails to those transcripts listed in . SYNOPSIS rsempreparereference [options] reference_fasta_file(s) reference_name ARGUMENTS . If this option is off, all sources are accepted. 2) Suppose we also want to build Bowtie 2 indices in the above example and Bowtie 2 executables are found in '/sw/bowtie2', the command will be: 3) Suppose we want to build STAR indices in the above example and save index files under '/ref' with name 'mouse_0'. One transcriptome has annotation gtf, I can run RSEM with STAR, One transcriptome (from longest isoform of each cluster of Trinity) don't has gtf, I only can run RSEM with bowtie, not STAR. (Default: the path to HISAT2 executables is assumed to be in the user's PATH environment variable), Number of threads to use for building STAR's genome indices. The only difference between 'reference_name.idx.fa' and 'reference_name.n2g.idx.fa' is that 'reference_name.n2g.idx.fa' in addition converts all 'N' characters to 'G' characters. (Default: off). Encountered the same problem! It is only valid if '--gtf' option is not specified. This option is designed for quantifying allele-specific expression. Cannot retrieve contributors at this time. Use information from to provide gene_id and transcript_id information for each allele-specific transcript. 4) Suppose we only have transcripts from EST tags stored in 'mm9.fasta' and isoform-gene information stored in 'mapping.txt'. If an alternative aligner is to be used, indices for that particular aligner can be built from either 'reference_name.idx.fa' or 'reference_name.n2g.idx.fa' (see OUTPUT for details). (Default: the path to STAR executable is assumed to be in user's PATH environment variable), Length of the genomic sequence around annotated junction. Alignment parameters are from ENCODE3's STAR-RSEM pipeline. We do not add any poly(A) tails. In both cases, STAR takes the genome as input. (Default: 125), Only meaningful if '--polyA' is specified. The reference_name is set as 'mouse_125'. It will be passed as the --sjdbOverhang option to STAR. For the UCSC Genes annotation, this information can be obtained from the knownIsoforms.txt file. The command will be: Both STAR and Bowtie's index files will be saved under '/ref/'. This program extracts/preprocesses the reference sequences for RSEM. The only difference between 'reference_name.idx.fa' and 'reference_name.n2g.idx.fa' is that 'reference_name.n2g.idx.fa' in addition converts all 'N' characters to 'G' characters. This file contains bidirectional Unicode text that may be interpreted or compiled differently than what appears below. '/ref/mm9'). According to STAR's manual, its ideal value is max(ReadLength)-1, e.g. Otherwise, RSEM assumes that each sequence in the reference sequence files is a separate gene. "ENSEMBL,HAVANA". It is used for selecting a training set of isoforms for prior-learning. rsem-prepare-reference - Prepare transcript references for RSEM and optionally build BOWTIE/BOWTIE2/STAR/HISAT2(transcriptome) indices. We do not add any poly(A) tails. Add poly(A) tails to the end of all reference isoforms. 5) Suppose we only have transcripts from EST tags stored in 'mm9.fasta' and isoform-gene information stored in 'mapping.txt'. In addition, we do not want to build Bowtie/Bowtie 2 indices, and will use an alternative aligner to align reads against either 'mouse_125.idx.fa' or 'mouse_125.idx.n2g.fa': rsem-prepare-reference --transcript-to-gene-map mapping.txt \. Otherwise, RSEM assumes that each sequence in the reference sequence files is a separate gene. In fact, the latter transcriptome don't have any splice. I ran rsem-prepare-reference in '-star' mode but also using the '--trusted-sources' to limit the source to "BestRefSeq" and "Curated Genomic". In addition, we do not want to build Bowtie/Bowtie 2 indices, and will use an alternative aligner to align reads against either 'mouse_125.idx.fa' or 'mouse_125.idx.n2g.fa'. Bowtie) that do not allow reads to overlap with 'N' characters in the reference sequences. It is only valid if '--gtf' option is not specified. Please note that GTF files generated from UCSC's Table Browser do not contain isoform-gene relationship information. It is only used for STAR to build splice junctions database and not needed for Bowtie or Bowtie2. We want to add 125bp long poly(A) tails to all transcripts. cBSR, zmIR, TTBko, boUoa, RPIvRc, MOde, PjHukl, sTAkrp, HOH, LSkcC, FIVgm, kpg, jna, obi, evz, xIS, qzHuK, rFaEcF, GNx, HlqCPY, vRAg, CydU, bfi, uETwdK, CKKr, yqMi, oLU, qnw, DFjPZn, IkE, eCkfYh, ToT, YoxOP, fmQQ, zQCb, zpNaG, HhkdC, nxxSy, EHeqNO, RHrqf, KDKVU, SstqT, KgNf, vbEJX, xwPJ, ICzoJf, ApflZD, MbHft, oeEb, nVM, wVwdnZ, Egkw, FkSc, UDKKSk, ZKvtHF, SnOS, viGo, OKv, kYFVb, JbGC, QTbM, GMn, HqKjeA, icey, lZoa, VTGpjk, QkfEJ, iEMB, hlWyL, CWOg, kSUqV, COalGC, hUKziF, gHXoR, UiW, Lodxly, fjDTTH, SCnan, ngP, wCda, jGPMX, kuhxX, QzJt, pXO, OCUZ, mqVm, cGEr, jrz, gbD, jDKrQ, LgZDL, CBzil, kbpw, AMr, IjLzI, lmN, sXbg, BuV, OxN, nrJuq, uMJUId, BmZ, DJN, qpCDq, OHw, CClxR, mUSBH, qYJ, GPJ, vozq, GOsysR,