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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 0e434c9b5c06 abundance_estimates_to_matrix.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/abundance_estimates_to_matrix.xml Fri Jul 31 08:06:22 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 0e434c9b5c06 align_and_estimate_abundance.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/align_and_estimate_abundance.xml Fri Jul 31 08:06:22 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 0e434c9b5c06 all_de_steps.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/all_de_steps.xml Fri Jul 31 08:06:22 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 0e434c9b5c06 blast_parser.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/blast_parser.xml Fri Jul 31 08:06:22 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 0e434c9b5c06 gtf2fasta.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/gtf2fasta.xml Fri Jul 31 08:06:22 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 0e434c9b5c06 insilico_read_normalization.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/insilico_read_normalization.xml Fri Jul 31 08:06:22 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 0e434c9b5c06 pfam_db.loc.sample --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/pfam_db.loc.sample Fri Jul 31 08:06:22 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 0e434c9b5c06 tool_dependencies.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/tool_dependencies.xml Fri Jul 31 08:06:22 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 0e434c9b5c06 transDecoder.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/transDecoder.xml Fri Jul 31 08:06:22 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 0e434c9b5c06 trinityStats.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/trinityStats.xml Fri Jul 31 08:06:22 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 0e434c9b5c06 trinityrnaseq.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/trinityrnaseq.xml Fri Jul 31 08:06:22 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 + +