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22 01:58:00 2015 -0400 @@ -0,0 +1,18 @@ +Introduction +============ + +Next Generation Sequence Analysis Pipelines (NGSAP) is to provide a user friendly platform for NGS analysis to support researchers working on myriad aspects of life sciences with post processing of the next generation sequencing data. It has beed designed specifically for transcriptomics tools available through Galaxy for data analysis which can be translated into meaningful scientific publications. NGSAP is an initiative of NGSAP team working in School of Computational and Integrative Sciences at Jawaharlal Nehru University, Delhi + +NGSAP provides two worlflows + + +Denovo Assembly and differential gene analysis workflow +======================================================== + +details of the workflow + + +Reference based assembly and differential gene analysis workflow +================================================================= +details of the workflow + diff -r 000000000000 -r cc71440b759f abundance_estimates_to_matrix.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/abundance_estimates_to_matrix.xml Tue Sep 22 01:58:00 2015 -0400 @@ -0,0 +1,39 @@ + + Generates combined Matrix for abundance estimates + + + trinityrnaseq + edgeR + TRINITY_HOME + + +\$TRINITY_HOME/util/abundance_estimates_to_matrix.pl + --est_method $est_method + #for $i in $isoform_sample: + $i.isoform_sample_matrix + #end for + > $output_log 2>&1; + + + + + + + + + + + + + + + + + + + + + + + + diff -r 000000000000 -r cc71440b759f align_and_estimate_abundance.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/align_and_estimate_abundance.xml Tue Sep 22 01:58:00 2015 -0400 @@ -0,0 +1,150 @@ + + +align_and_estimate_abundance + + + trinityrnaseq + rsem + eXpress + bowtie2 + bowtie + samtools + TRINITY_HOME + + +perl \$TRINITY_HOME/util/align_and_estimate_abundance.pl + --transcripts $transcripts + --est_method $est_method + --aln_method $aln_method + --prep_reference + + ## Inputs. + #if str($reads.paired_or_single) == "paired": + + --left $reads.left_input --right $reads.right_input + + #if $reads.left_input.ext == 'fa': + --seqType fa + #else: + --seqType fq + #end if + + ## Additional parameters. + #if str($reads.optional.use_options) == "yes": + + #if str($reads.optional.library_type) != "None": + --SS_lib_type $reads.optional.library_type + #end if + + + + #end if + + #else: + --single $reads.input + + #if str($reads.input.ext) == 'fa': + --seqType fa + #else: + --seqType fq + #end if + + ## Additional parameters. + #if str($reads.optional.use_option) == "yes": + + #if str($reads.additional_params.library_type) != "None": + --SS_lib_type $reads.optional.library_type + #end if + + #end if + #end if + +## direct to output +> $align_and_estimate_abundance_log 2>&1;samtools sort bowtie.bam bowtie_sorted + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + diff -r 000000000000 -r cc71440b759f all_de_steps.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/all_de_steps.xml Tue Sep 22 01:58:00 2015 -0400 @@ -0,0 +1,94 @@ + +Generates results for DE + + + trinityrnaseq + TRINITY_HOME + + + + +echo -e "Differential Expression Log File" > $output_log 2>&1; + +## Step 0: preprocess input_file preparaton + + #for $i in $isoform_sample: + cp -s $i.isoform_sample_matrix $i.sample_name; + #end for; + + +## Step 1: abundance_estimates_to_matrix +echo -e "" >> $output_log 2>&1; +echo -e "\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#" >> $output_log 2>&1; +echo -e "\#\# Step 1: abundance_estimates_to_matrix" >> $output_log 2>&1; +echo -e "" >> $output_log 2>&1; + + +perl \$TRINITY_HOME/util/abundance_estimates_to_matrix.pl + --est_method $est_method + #for $i in $isoform_sample: + $i.sample_name + #end for + >> $output_log 2>&1; + + +## Step 2: run_DE_analysis +echo -e "" >> $output_log 2>&1; +echo -e "\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#" >> $output_log 2>&1; +echo -e "\#\# Step 2: run_DE_analysis" >> $output_log 2>&1; +echo -e "" >> $output_log 2>&1; + +perl \$TRINITY_HOME/Analysis/DifferentialExpression/run_DE_analysis.pl + --matrix matrix.counts.matrix + --method $method + --output result_dir + >> $output_log 2>&1; + +## Step 3: analyze_diff_expr +echo -e "" >> $output_log 2>&1; +echo -e "\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#" >> $output_log 2>&1; +echo -e "\#\# Step 3: analyze_diff_expr" >> $output_log 2>&1; +echo -e "" >> $output_log 2>&1; + +cd result_dir && perl \$TRINITY_HOME/Analysis/DifferentialExpression/analyze_diff_expr.pl + + --matrix ../matrix.TMM.fpkm.matrix + + >> $output_log 2>&1; + +pwd; +cd .. && zip -r results.zip result_dir; + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + diff -r 000000000000 -r cc71440b759f blast_parser.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/blast_parser.xml Tue Sep 22 01:58:00 2015 -0400 @@ -0,0 +1,59 @@ + + + +parses Blast Output File + + + /bin/awk + 'BEGIN {FS="\t"} {if($11 <= $evalue && $13 >= $qcovs) print $0 }' + $blastoutfile + > $parsed_blastfile_above_cutoff; + + /bin/awk + '{FS="\t"} {if($11 > $evalue || $13 < $qcovs) print $0 }' + $blastoutfile + > $parsed_blastfile_below_cutoff; + + /bin/sed -e '/^>/s/$/@@@@/' -e 's/^>/#/' $trinityassembledfile + | tr -d '\n' + | tr "#" '\n' + | sed -e 's/^/>/' -e 's/@@@@/\n/' + | sed -e "1d" + > $oneline_file; + + /bin/awk + 'BEGIN {FS="|"} {print "grep -w -A 1 \""$1"\" $oneline_file >> $blastfile_below_cutoff"}' + $parsed_blastfile_below_cutoff + > pick_below_sh; + + /bin/sh pick_below_sh; + + /bin/awk + 'BEGIN {FS="|"} {print "grep -w -A 1 \""$1"\" $oneline_file >> $blastfile_above_cutoff"}' + $parsed_blastfile_above_cutoff + > pick_above_sh; + + /bin/sh pick_above_sh; + + + + + + + + + + + + + + + + + + + +blast output parser tool + + + diff -r 000000000000 -r cc71440b759f gtf2fasta.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/gtf2fasta.xml Tue Sep 22 01:58:00 2015 -0400 @@ -0,0 +1,30 @@ + + + + Converts gtf to fasta + + + tophat2 + + +gtf_to_fasta $gtf $genome $fasta + + + + + + + + + + + + + +**What it does** + Converts gtf file to fasta using reference genome + + + + + diff -r 000000000000 -r cc71440b759f insilico_read_normalization.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/insilico_read_normalization.xml Tue Sep 22 01:58:00 2015 -0400 @@ -0,0 +1,152 @@ + + + +Read Normalization + + trinityrnaseq + TRINITY_HOME + + + \$TRINITY_HOME/util/insilico_read_normalization.pl +--JM $JM +--max_cov $maximum_coverage + +## Inputs. +#if str($inputs.paired_or_single) == "paired": + + --left $inputs.left_input --right $inputs.right_input + + #if $inputs.left_input.ext == 'fa': + --seqType fa + #else: + --seqType fq + #end if + + ## Additional parameters. + #if str($inputs.additional_params.use_additional) == "yes": + + #if str($inputs.additional_params.library_type) != "None": + --SS_lib_type $inputs.additional_params.library_type + #end if + + $inputs.additional_params.pairs_together + --CPU $inputs.additional_params.CPU + $inputs.additional_params.PARALLEL_STATS + --KMER_SIZE $inputs.additional_params.KMER_SIZE + --max_pct_stdev $inputs.additional_params.max_pct_stdev + + #end if + + +#else: + + --single $inputs.input + + #if str($inputs.input.ext) == 'fa': + --seqType fa + #else: + --seqType fq + #end if + + ## Additional parameters. + #if str($inputs.additional_params.use_additional) == "yes": + + #if str($inputs.additional_params.library_type) != "None": + --SS_lib_type $inputs.additional_params.library_type + #end if + + ##$inputs.additional_params.pairs_together + --CPU $inputs.additional_params.CPU + ##$inputs.additional_params.PARALLEL_STATS + --KMER_SIZE $inputs.additional_params.KMER_SIZE + --max_pct_stdev $inputs.additional_params.max_pct_stdev + + #end if + +#end if + + + +## direct to output +> $insilico_read_normalization_log 2>&1 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +Insilico Read Normalization + + diff -r 000000000000 -r cc71440b759f pfam_db.loc.sample --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/pfam_db.loc.sample Tue Sep 22 01:58:00 2015 -0400 @@ -0,0 +1,3 @@ + +###1_name ###2_date ###2_path_to_db +pfam_db.hmm july 2014 /home/username/galaxy-dist/tool-data/pfam_db.hmm diff -r 000000000000 -r cc71440b759f tool_dependencies.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/tool_dependencies.xml Tue Sep 22 01:58:00 2015 -0400 @@ -0,0 +1,336 @@ + + + + + + http://ngsaptools.osdd.jnu.ac.in/ngsap/trinityrnaseq_r20140717.tar.gz + make + + . + $INSTALL_DIR + + + $INSTALL_DIR + + + $INSTALL_DIR + + + + + + + + + + + http://depot.galaxyproject.org/package/linux/i386/samtools/samtools-0.1.18-linux-i386.tgz + + . + $INSTALL_DIR + + + + http://depot.galaxyproject.org/package/linux/x86_64/samtools/samtools-0.1.18-linux-x86_64.tgz + + . + $INSTALL_DIR + + + + http://depot.galaxyproject.org/package/darwin/i386/samtools/samtools-0.1.18-Darwin-i386.tgz + + . + $INSTALL_DIR + + + + http://depot.galaxyproject.org/package/darwin/x86_64/samtools/samtools-0.1.18-Darwin-x86_64.tgz + + . + $INSTALL_DIR + + + + http://depot.galaxyproject.org/package/source/samtools/samtools-0.1.18.tar.bz2 + sed -i.bak 's/-lcurses/-lncurses/' Makefile + make + + samtools + $INSTALL_DIR/bin + + + libbam.a + $INSTALL_DIR/lib + + + . + $INSTALL_DIR/include/bam + + + + $INSTALL_DIR/bin + $INSTALL_DIR/lib + $INSTALL_DIR + + + + + + + + + + http://deweylab.biostat.wisc.edu/rsem/src/rsem-1.1.17.tar.gz + make + $INSTALL_DIR/bin/sam + + sam/samtools + $INSTALL_DIR/bin/sam + + + convert-sam-for-rsem + $INSTALL_DIR/bin + + + extract-transcript-to-gene-map-from-trinity + $INSTALL_DIR/bin + + + rsem-bam2readdepth + $INSTALL_DIR/bin + + + rsem-bam2wig + $INSTALL_DIR/bin + + + rsem-build-read-index + $INSTALL_DIR/bin + + + rsem-calculate-credibility-intervals + $INSTALL_DIR/bin + + + rsem-calculate-expression + $INSTALL_DIR/bin + + + rsem-extract-reference-transcripts + $INSTALL_DIR/bin + + + rsem-gen-transcript-plots + $INSTALL_DIR/bin + + + rsem-get-unique + $INSTALL_DIR/bin + + + rsem-parse-alignments + $INSTALL_DIR/bin + + + rsem-plot-model + $INSTALL_DIR/bin + + + rsem-plot-transcript-wiggles + $INSTALL_DIR/bin + + + rsem-prepare-reference + $INSTALL_DIR/bin + + + rsem-preref + $INSTALL_DIR/bin + + + rsem-run-em + $INSTALL_DIR/bin + + + rsem-run-gibbs + $INSTALL_DIR/bin + + + rsem-simulate-reads + $INSTALL_DIR/bin + + + rsem-synthesis-reference-transcripts + $INSTALL_DIR/bin + + + rsem-tbam2gbam + $INSTALL_DIR/bin + + + $INSTALL_DIR/bin + + + + + + + + + + + http://bio.math.berkeley.edu/eXpress/downloads/express-1.5.1/express-1.5.1-linux_x86_64.tgz + + . + $INSTALL_DIR/bin + + + + http://bio.math.berkeley.edu/eXpress/downloads/express-1.5.1/express-1.5.1-macosx_x86_64.tgz + + . + $INSTALL_DIR/bin + + + + http://bio.math.berkeley.edu/eXpress/downloads/express-1.5.1/express-1.5.1-src.tgz + cmake src -DCMAKE_INSTALL_PREFIX:PATH=$INSTALL_DIR + make && make install + + + $INSTALL_DIR/bin + + + + + + + + + + http://depot.galaxyproject.org/package/linux/x86_64/bowtie/bowtie-0.12.7-linux-x86_64.tgz + + . + $INSTALL_DIR + + + + http://depot.galaxyproject.org/package/darwin/x86_64/bowtie/bowtie-0.12.7-macos-10.5-x86_64.tgz + + . + $INSTALL_DIR + + + + http://depot.galaxyproject.org/package/linux/i386/bowtie/bowtie-0.12.7-linux-i386.tgz + + . + $INSTALL_DIR + + + + http://depot.galaxyproject.org/package/darwin/i386/bowtie/bowtie-0.12.7-macos-10.5-i386.tgz + + . + $INSTALL_DIR + + + + http://downloads.sourceforge.net/project/bowtie-bio/bowtie/0.12.7/bowtie-0.12.7-src.zip + make + + bowtie + $INSTALL_DIR + + + bowtie-build + $INSTALL_DIR + + + bowtie-inspect + $INSTALL_DIR + + + + $INSTALL_DIR + + + + + + + + + + http://depot.galaxyproject.org/package/linux/x86_64/bowtie2/bowtie2-2.1.0-linux-x86_64.tgz + + . + $INSTALL_DIR + + + + http://depot.galaxyproject.org/package/darwin/x86_64/bowtie2/bowtie2-2.1.0-macos-x86_64.tgz + + . + $INSTALL_DIR + + + + http://depot.galaxyproject.org/package/linux/i386/bowtie2/bowtie2-2.1.0-linux-i386.tgz + + . + $INSTALL_DIR + + + + http://depot.galaxyproject.org/package/darwin/i386/bowtie2/bowtie2-2.1.0-macos-i386.tgz + + . + $INSTALL_DIR + + + + http://downloads.sourceforge.net/project/bowtie-bio/bowtie2/2.1.0/bowtie2-2.1.0-source.zip + make + + bowtie2 + $INSTALL_DIR + + + bowtie2-align + $INSTALL_DIR + + + bowtie2-build + $INSTALL_DIR + + + bowtie2-inspect + $INSTALL_DIR + + + + $INSTALL_DIR + + + + + + + + + + + + + + + + + + + + + + diff -r 000000000000 -r cc71440b759f transDecoder.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/transDecoder.xml Tue Sep 22 01:58:00 2015 -0400 @@ -0,0 +1,120 @@ + + coding region + + + trinityrnaseq + TRINITY_HOME + + +perl \$TRINITY_HOME/trinity-plugins/transdecoder/TransDecoder + + -t $transcript + + #if str($optional.parameters) == 'yes': + #if ($train): + --train $train + #end if + + #if ($min_protein_length): + -m $min_protein_length + #end if + + #if ($genetic_code): + -G $genetic_code + #end if + + #if ($strand-specific): + $strand-specific + #end if + + #if ($lottmn): + -T $lottmm + #end if + + #if ($retain_long_orfs): + --retain_long_orfs $retain_long_orfs + #end if + #end if + + #if str($pfam.options) == 'yes': + #if ($pfam_db): + --search_pfam $pfam_db + #end if + + #if ($CPU): + --CPU $CPU + #end if + #end if + + ##log file + > $transdecoder_log 2>&1; + + + ## output renaming + + cp -s dataset_*.dat.transdecoder.pep transcript.transdecoder.pep; + cp -s dataset_*.dat.transdecoder.cds transcript.transdecoder.cds; + cp -s dataset_*.dat.transdecoder.bed transcript.transdecoder.bed; + cp -s dataset_*.dat.transdecoder.gff3 transcript.transdecoder.gff3; + cp -s dataset_*.dat.transdecoder.mRNA transcript.transdecoder.mRNA; + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + diff -r 000000000000 -r cc71440b759f trinityStats.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/trinityStats.xml Tue Sep 22 01:58:00 2015 -0400 @@ -0,0 +1,25 @@ + + check stats... + + + trinityrnaseq + TRINITY_HOME + + +perl \$TRINITY_HOME/util/TrinityStats.pl $fastafile +> $statsfile 2>&1; + + + + + + + + + + + + + + + diff -r 000000000000 -r cc71440b759f trinityrnaseq.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/trinityrnaseq.xml Tue Sep 22 01:58:00 2015 -0400 @@ -0,0 +1,131 @@ + + + + De novo assembly of RNA-Seq data Using Trinity + + trinityrnaseq + samtools + bowtie + TRINITY_HOME + + + + \$TRINITY_HOME/Trinity --JM $JM --CPU $CPU + + ## Inputs. + #if str($inputs.paired_or_single) == "paired": + --left $inputs.left_input --right $inputs.right_input + #if $inputs.left_input.ext == 'fa': + --seqType fa + #else: + --seqType fq + #end if + #if str($inputs.library_type) != "None": + --SS_lib_type $inputs.library_type + #end if + --group_pairs_distance $inputs.group_pairs_distance + #else: + --single $inputs.input + #if str($inputs.input.ext) == 'fa': + --seqType fa + #else: + --seqType fq + #end if + #if str($inputs.library_type) != "None": + --SS_lib_type $inputs.library_type + #end if + #end if + + ## Additional parameters. + #if str($additional_params.use_additional) == "yes": + --min_kmer_cov $inputs.min_kmer_cov --max_reads_per_graph $inputs.max_reads_per_graph --bflyHeapSpaceMax $input.bflyHeapSpaceMax + #if $inputs.bfly_opts != 'None': + --bfly_opts " $inputs.bfly_opts " + #end if + #end if + + + ## direct to output + > $trinity_log 2>&1 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Trinity is a de novo transcript assembler that uses RNA-seq data as input. This tool runs all Trinity_ commands--Inchworm, Chrysalis, and Butterfly--in a single pass. + + .. _Trinity: http://trinityrnaseq.sourceforge.net + + diff -r 000000000000 -r cc71440b759f trinityrnaseq.xml~ --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/trinityrnaseq.xml~ Tue Sep 22 01:58:00 2015 -0400 @@ -0,0 +1,131 @@ + + + + De novo assembly of RNA-Seq data Using Trinity + + trinityrnaseq + samtools + bowtie + TRINITY_HOME + + + + \$TRINITY_HOME/Trinity --JM $JM --CPU $CPU + + ## Inputs. + #if str($inputs.paired_or_single) == "paired": + --left $inputs.left_input --right $inputs.right_input + #if $inputs.left_input.ext == 'fa': + --seqType fa + #else: + --seqType fq + #end if + #if str($inputs.library_type) != "None": + --SS_lib_type $inputs.library_type + #end if + --group_pairs_distance $inputs.group_pairs_distance + #else: + --single $inputs.input + #if str($inputs.input.ext) == 'fa': + --seqType fa + #else: + --seqType fq + #end if + #if str($inputs.library_type) != "None": + --SS_lib_type $inputs.library_type + #end if + #end if + + ## Additional parameters. + #if str($additional_params.use_additional) == "yes": + --min_kmer_cov $inputs.min_kmer_cov --max_reads_per_graph $inputs.max_reads_per_graph --bflyHeapSpaceMax $input.bflyHeapSpaceMax + #if $inputs.bfly_opts != 'None': + --bfly_opts " $inputs.bfly_opts " + #end if + #end if + + + ## direct to output + > $trinity_log 2>&1 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Trinity is a de novo transcript assembler that uses RNA-seq data as input. This tool runs all Trinity_ commands--Inchworm, Chrysalis, and Butterfly--in a single pass. + + .. _Trinity: http://trinityrnaseq.sourceforge.net + +