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Our mission is to provide a user friendly platform for NGS analysis to support Indian researchers working on myriad aspects of life sciences with post processing of the next generation sequencing data. Present portal introduces readers to the 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 + +Two workflows in NGSAP +======================== + +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 d4c781f09244 abundance_estimates_to_matrix.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/abundance_estimates_to_matrix.xml Fri Jul 31 09:33:03 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 d4c781f09244 align_and_estimate_abundance.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/align_and_estimate_abundance.xml Fri Jul 31 09:33:03 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 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + diff -r 000000000000 -r d4c781f09244 all_de_steps.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/all_de_steps.xml Fri Jul 31 09:33:03 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 d4c781f09244 blast_parser.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/blast_parser.xml Fri Jul 31 09:33:03 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 d4c781f09244 gtf2fasta.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/gtf2fasta.xml Fri Jul 31 09:33:03 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 d4c781f09244 insilico_read_normalization.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/insilico_read_normalization.xml Fri Jul 31 09:33:03 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 d4c781f09244 pfam_db.loc.sample --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/pfam_db.loc.sample Fri Jul 31 09:33:03 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 d4c781f09244 tool_dependencies.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/tool_dependencies.xml Fri Jul 31 09:33:03 2015 -0400 @@ -0,0 +1,333 @@ + + + + + + http://tools.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 d4c781f09244 transDecoder.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/transDecoder.xml Fri Jul 31 09:33:03 2015 -0400 @@ -0,0 +1,120 @@ + + coding region + + + trinityrnaseq + TRINITY_HOME + + + \$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 d4c781f09244 trinityStats.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/trinityStats.xml Fri Jul 31 09:33:03 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 d4c781f09244 trinityrnaseq.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/trinityrnaseq.xml Fri Jul 31 09:33:03 2015 -0400 @@ -0,0 +1,129 @@ + + + + De novo assembly of RNA-Seq data Using Trinity + + trinityrnaseq + 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 + +