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/*
* Copyright 2013 Brian Tjaden
*
* This file is part of Rockhopper.
*
* Rockhopper is free software: you can redistribute it and/or modify
* it under the terms of the GNU General Public License as published by
* the Free Software Foundation, either version 3 of the License, or
* any later version.
*
* Rockhopper is distributed in the hope that it will be useful,
* but WITHOUT ANY WARRANTY; without even the implied warranty of
* MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
* GNU General Public License for more details.
*
* You should have received a copy of the GNU General Public License
* (in the file gpl.txt) along with Rockhopper.
* If not, see <http://www.gnu.org/licenses/>.
*/
import java.util.ArrayList;
import java.io.File;
import java.io.FileNotFoundException;
import java.io.PrintWriter;
import javax.swing.JTextArea;
import javax.swing.SwingWorker;
/**
* The Rockhopper application reads in RNA-seq data, aligns the
* sequencing reads to a genome, and then analyzes the data to
* identify the expression of genes in each condition, differential
* gene expression between conditions, transcript boundaries, and
* operons. Rockhopper can also assemble transcripts de novo
* without a reference genome.
*/
public class Rockhopper {
/*****************************************
********** CLASS VARIABLES **********
*****************************************/
public static final String version = "2.03";
public static JTextArea output; // Output to GUI or, if null, to System.out
public static SwingWorker worker; // Update progress bar if using GUI.
private static int stagesProcessed; // Number of files (stages) processed. Needed for GUI progress bar.
public static ArrayList<Integer> genomeSizes; // Sizes of each 1-indexed genome in nucleotides
public static int genomeSize; // Size of all genomes combined
public static int numConditions; // Number of experimental conditions
private static ArrayList<String> genome_DIRs; // PARAM: directories of geneome files
public static String output_DIR = "Rockhopper_Results/"; // PARAM: write output files to this directory
public static String browser_DIR = "genomeBrowserFiles/"; // Write browser files to this sub-directory
private static ArrayList<String> conditionFiles; // PARAM: list of seq-read files for each condition
public static boolean computeExpression = true; // PARAM: compute differential expression
public static boolean computeOperons = true; // PARAM: compute operons
public static boolean computeTranscripts = true; // PARAM: compute transcript boundaries
private static PrintWriter summaryWriter = null; // For outputting summary file
private static String summaryFile = "summary.txt";
private static String expressionFile = "transcripts.txt";
private static String operonGenePairFile = "operonGenePairs.txt";
private static String operonMergedFile = "operons.txt";
public static boolean unstranded = false; // Is RNA-seq data strand specific or ambiguous?
public static double transcriptSensitivity = 0.5;
public static boolean verbose = false;
private static String[] labels;
private static boolean time = false;
private static boolean isDeNovo = false; // Reference based (false) or de novo (true) assembly
/********************************************
********** INSTANCE VARIABLES **********
********************************************/
private ArrayList<Genome> genomes = new ArrayList<Genome>();
private ArrayList<Transcripts> transcripts = new ArrayList<Transcripts>();
private ArrayList<Condition> conditions; // RNA-seq data for each condition
/**************************************
********** CONSTRUCTORS **********
**************************************/
/**
* Constructs a new Rockhopper object.
*/
public Rockhopper() {
stagesProcessed = 0;
if (isDeNovo) { // De novo assembly
Assembler a = new Assembler();
return;
}
// Read in genome
Rockhopper.genomeSizes = new ArrayList<Integer>();
Rockhopper.genomeSize = 0;
for (String genome_DIR : genome_DIRs) {
Genome genome = new Genome(genome_DIR);
this.genomes.add(genome);
Rockhopper.genomeSizes.add(genome.size());
Rockhopper.genomeSize += genome.size();
}
// Align sequencing reads to genome and compute basic expression information
this.conditions = new ArrayList<Condition>();
Peregrine.sequenceFiles = new String[genomes.size()];
Peregrine.annotationsPlus = new String[genomes.size()][];
Peregrine.annotationsMinus = new String[genomes.size()][];
for (int z=0; z<genomes.size(); z++) {
String genome_DIR = genome_DIRs.get(z);
File dir = new File(genome_DIR);
String[] files = dir.list();
for (int i=0; i<files.length; i++) {
if (files[i].endsWith(".fna")) {
Peregrine.sequenceFiles[z] = genome_DIR + files[i];
Peregrine.annotationsPlus[z] = genomes.get(z).getAnnotations('+');
Peregrine.annotationsMinus[z] = genomes.get(z).getAnnotations('-');
}
}
}
Peregrine.numSequences = genomes.size();
Peregrine.outputBrowserFile = false;
output("\n");
for (int i=0; i<conditionFiles.size(); i++) { // For each condition
String[] readFiles = conditionFiles.get(i).split(",");
Condition C = new Condition();
for (int j=0; j<readFiles.length; j++) { // For each replicate
String[] parse_readFiles = readFiles[j].split("%");
if (parse_readFiles.length == 1) { // Using single-end reads (except maybe SAM/BAM)
String file1 = parse_readFiles[0];
output("Aligning sequencing reads from file:\t" + file1.split(separator())[file1.split(separator()).length-1] + "\n");
Peregrine.isPairedEnd = false;
Peregrine.pairedEndFile = null;
Peregrine.readsFile = file1;
Peregrine p = new Peregrine();
updateProgress();
C.addReplicate(new Replicate(p.getListOfCompressedFileNames(file1), file1, unstranded));
} else { // Using paired-end reads
String file1 = parse_readFiles[0];
String file2 = parse_readFiles[1];
output("Aligning sequencing reads from files:\n");
output("\t" + file1.split(separator())[file1.split(separator()).length-1] + "\n");
output("\t" + file2.split(separator())[file2.split(separator()).length-1] + "\n");
Peregrine.isPairedEnd = true;
Peregrine.readsFile = file1;
Peregrine.pairedEndFile = file2;
Peregrine p = new Peregrine();
updateProgress();
C.addReplicate(new Replicate(p.getListOfCompressedFileNames(file1), file1, unstranded));
}
}
C.setMinDiffExpressionLevel();
conditions.add(C);
}
Rockhopper.numConditions = conditions.size();
computeGeneExpression();
// Output mapped reads to WIG files that can be loaded by genome browser
double trackRange = 0.0;
int totalReplicates = 0;
for (int i=0; i<conditions.size(); i++) {
for (int j=0; j<conditions.get(i).numReplicates(); j++) {
trackRange += conditions.get(i).getReplicate(j).getAvgReads();
totalReplicates++;
}
}
trackRange = (2*trackRange) / totalReplicates; // Range for display in genome browser
for (int i=0; i<conditionFiles.size(); i++) { // For each condition
String[] readFiles = conditionFiles.get(i).split(",");
for (int j=0; j<readFiles.length; j++) { // For each replicate
Replicate r = conditions.get(i).getReplicate(j);
double multiplier = 100000.0 / (double)r.getUpperQuartile();
for (int z=0; z<genomes.size(); z++) {
int[] coordinates_plus = new int[Rockhopper.genomeSizes.get(z)];
int[] coordinates_minus = new int[Rockhopper.genomeSizes.get(z)];
for (int k=0; k<Rockhopper.genomeSizes.get(z); k++) {
coordinates_plus[k] = (int)(multiplier * r.getReads(z, k, '+'));
coordinates_minus[k] = (int)(multiplier * r.getReads(z, k, '-'));
}
Peregrine.outputBrowserFile(readFiles[j].split("%")[0], Peregrine.sequenceFiles[z], output_DIR, browser_DIR, r.getCompressedFileName(z), coordinates_plus, coordinates_minus, (int)trackRange);
}
}
}
Peregrine.releaseMemory();
output("Analyzing transcripts..." + "\n");
// Compute transcript boundaries
if (computeTranscripts) {
for (int z=0; z<genomes.size(); z++) {
Genome genome = genomes.get(z);
if (genome.numGenes() > 0) {
Transcripts t = new Transcripts(z, genome, conditions, unstranded);
t.identifyUTRs();
t.identifyRNAs();
transcripts.add(t);
}
}
}
// Compute gene differential expression
if (computeExpression) {
Gene.setLowessVariances(genomes, conditions); // Compute lowess variance for each gene
for (Genome genome : genomes) {
if (genome.numGenes() > 0) computeDifferentialExpression(genome);
}
Gene.correctPvalues(genomes, conditions); // Compute q-values, Benjamini-Hochberg correction
}
for (int z=0; z<genomes.size(); z++) runRockhopper(z);
output("\nSummary of results written to file:\t" + output_DIR + summaryFile + "\n");
for (int z=0; z<genomes.size(); z++) {
output("Expression of transcripts written to file:\t" + output_DIR + genomes.get(z).getID() + "_" + expressionFile + "\n");
if (computeOperons) {
//output("Pairs of operon genes written to file:\t" + output_DIR + genome.getID() + "_" + operonGenePairFile + "\n");
output("Operons written to file:\t\t" + output_DIR + genomes.get(z).getID() + "_" + operonMergedFile + "\n");
}
}
output("\nFINISHED.\n\n");
summaryWriter.close();
updateProgress();
}
/*************************************************
********** PUBLIC INSTANCE METHODS **********
*************************************************/
public void runRockhopper(int z) {
Genome genome = genomes.get(z);
output("\n");
output("************************************************************\n");
output(genome.getName() + "\n");
output("************************************************************\n");
output("\n");
// Compute transcript boundaries
if (computeTranscripts && (genome.numGenes() > 0)) {
Transcripts t = transcripts.get(z);
output("Computing transcript boundaries...\n");
output("\tNumber of 5'UTRs:\t\t\t" + t.getNum5UTRs() + "\n");
output("\tNumber of 3'UTRs:\t\t\t" + t.getNum3UTRs() + "\n");
output("\tNumber of predicted RNAs:\t\t\t" + (t.getNumSenseRNAs() + t.getNumAntisenseRNAs()) + "\n");
if (!unstranded) {
output("\tNumber of predicted RNAs (not antisense):\t\t" + t.getNumSenseRNAs() + "\n");
output("\tNumber of predicted RNAs (antisense):\t\t" + t.getNumAntisenseRNAs() + "\n");
}
output("\n");
outputUTRsForBrowser(output_DIR + browser_DIR + genome.getID() + "_UTRs.wig", genome.getFormalGenomeName(), genome.getGenes(), genome.size());
outputRNAsForBrowser(output_DIR + browser_DIR + genome.getID() + "_ncRNAs.wig", genome.getFormalGenomeName(), genome.getGenes(), genome.size());
}
// Compute gene differential expression
if (computeExpression && (genome.numGenes() > 0)) {
output("Computing differential gene expression...\n");
output("\tNumber of differentially expressed protein coding genes:\t" + getNumOfDifferentiallyExpressedGenes(genome, 0.01) + "\n\n");
outputDifferentiallyExpressedGenesForBrowser(output_DIR + browser_DIR + genome.getID() + "_diffExpressedGenes.wig", genome.getFormalGenomeName(), genome.getGenes(), genome.size());
}
// Output transcript file
try {
PrintWriter writer = new PrintWriter(new File(output_DIR + genome.getID() + "_" + expressionFile));
writer.println(genome.genesToString(conditions, labels));
writer.close();
} catch (FileNotFoundException e) {
output("\nError - could not open file " + genome.getID() + "_" + expressionFile + "\n\n");
}
// Compute operons
if (computeOperons && (genome.numGenes() > 0)) {
output("Computing likely operons...\n");
Operons ops = new Operons(genome.getCodingGenes(), genome.getGenes());
output("\tNumber of gene-pairs predicted to be part of the same operon:\t" + ops.getNumOperonGenePairs(genome.getCodingGenes()) + "\n");
//ops.outputGenePairOperons(output_DIR + genome.getID() + "_" + operonGenePairFile, genome.getCodingGenes());
int numMergedOperons = ops.outputMergedOperons(output_DIR + genome.getID() + "_" + operonMergedFile, output_DIR + browser_DIR + genome.getID() + "_operons.wig", genome.getFormalGenomeName(), genome.getCodingGenes(), genome.size());
output("\tNumber of predicted multi-gene operons:\t\t" + numMergedOperons + "\n\n");
}
}
/**
* Returns a list of Genome objects.
*/
public ArrayList<Genome> getGenomes() {
return genomes;
}
/**************************************************
********** PRIVATE INSTANCE METHODS **********
**************************************************/
/**
* For each gene, compute its raw read counts, normalized read
* counts, mean, variance, and RPKM.
*/
private void computeGeneExpression() {
int totalReplicates = 0;
long sumUpperQuartile = 0;
for (int j=0; j<conditions.size(); j++) {
for (int k=0; k<conditions.get(j).numReplicates(); k++) {
Replicate r = conditions.get(j).getReplicate(k);
r.setMinExpression(transcriptSensitivity);
ArrayList<Long> currentGeneReads = new ArrayList<Long>(); // For normalization
int zeroCountGenes = 0; // For normalization, keep track of genes with zero reads
int totalGenes = 0;
for (int z=0; z<genomes.size(); z++) {
Genome genome = genomes.get(z);
for (int i=0; i<genome.numGenes(); i++) {
Gene g = genome.getGene(i);
long readsForGene = r.getReadsInRange(z, g.getMinCoordinate(), g.getMaxCoordinate(), g.getStrand());
if (unstranded) // Strand ambiguous. Count reads on both strands.
readsForGene = r.getReadsInRange(z, g.getMinCoordinate(), g.getMaxCoordinate(), '+') + r.getReadsInRange(z, g.getMinCoordinate(), g.getMaxCoordinate(), '-');
g.setRawCount(j, k, readsForGene); // Set raw counts for gene
if (r.getAvgLengthReads() == 0) g.setRawCount_reads(j, k, 0);
else g.setRawCount_reads(j, k, readsForGene/r.getAvgLengthReads());
currentGeneReads.add(readsForGene); // Populate list for normalization
if (readsForGene == 0) zeroCountGenes++; // Genes with zero reads for normalization
}
totalGenes += genome.numGenes();
}
long upperQuartile = Misc.select_Long(currentGeneReads, (int)(zeroCountGenes+0.75*(totalGenes-zeroCountGenes)));
r.setUpperQuartile(upperQuartile);
sumUpperQuartile += upperQuartile;
totalReplicates++;
}
}
Condition.setAvgUpperQuartile(sumUpperQuartile / totalReplicates);
// Set normalized read counts for each gene
for (int j=0; j<conditions.size(); j++) {
for (int k=0; k<conditions.get(j).numReplicates(); k++) {
for (int z=0; z<genomes.size(); z++) {
Genome genome = genomes.get(z);
for (int i=0; i<genome.numGenes(); i++) { // Set normalized counts
genome.getGene(i).setNormalizedCount(j, k, 100000.0, conditions.get(j).getReplicate(k).getUpperQuartile());
}
}
}
}
// Compute mean and RPKM for each gene in each condition
for (int z=0; z<genomes.size(); z++) {
Genome genome = genomes.get(z);
for (int i=0; i<genome.numGenes(); i++) genome.getGene(i).computeExpression(conditions);
}
identifySimilarConditions(); // We require means to identify similar conditions
// Compute variance for each gene in each condition
for (int z=0; z<genomes.size(); z++) {
Genome genome = genomes.get(z);
for (int i=0; i<genome.numGenes(); i++) genome.getGene(i).computeVariance(conditions);
}
}
private void computeDifferentialExpression(Genome genome) {
// Compute differential expression for each gene in pairs of conditions
for (int i=0; i<genome.numGenes(); i++) genome.getGene(i).computeDifferentialExpression();
}
/**
* Returns the number of significantly differentially expressed
* protein-coding genes.
* The significance threshold is specified by the parameter.
*/
private int getNumOfDifferentiallyExpressedGenes(Genome genome, double significance) {
int numDiffExpressedGenes = 0;
for (int i=0; i<genome.numGenes(); i++) {
if (genome.getGene(i).isDifferntiallyExpressedORF(significance))
numDiffExpressedGenes++;
}
return numDiffExpressedGenes;
}
/**
* For each condition, determines the other condition that is most similar,
* i.e., its "partner". If there are no replicate experiments then the
* partner of each condition is used as a surrogate replicate.
* Similarity between two conditions is measured by the Pearson correlation
* coefficient of normalized gene expression.
* This method sets the "partner" of each condition.
*/
private void identifySimilarConditions() {
if (conditions.size() == 1) { // Special case. Only 1 condition. It partners itself.
conditions.get(0).setPartner(0);
return;
}
double[][] correlationMatrix = new double[conditions.size()][conditions.size()];
for (int i=0; i<conditions.size(); i++) {
for (int j=0; j<conditions.size(); j++) {
ArrayList<Long> e1 = new ArrayList<Long>(); // Gene expression values 1
ArrayList<Long> e2 = new ArrayList<Long>(); // Gene expression values 2
for (Genome genome : genomes) {
for (int k=0; k<genome.numGenes(); k++) {
int geneLength = genome.getGene(k).getMaxCoordinate() - genome.getGene(k).getMinCoordinate() + 1;
e1.add(genome.getGene(k).getMean(i)/geneLength);
e2.add(genome.getGene(k).getMean(j)/geneLength);
}
}
correlationMatrix[i][j] = Misc.correlation(e1, e2);
}
}
// Determine partner for each condition
int partner = -1;
for (int i=0; i<conditions.size(); i++) {
if (i == 0) partner = 1;
else partner = 0;
for (int j=0; j<conditions.size(); j++) {
if ((i != j) && (correlationMatrix[i][j] > correlationMatrix[i][partner]))
partner = j;
}
conditions.get(i).setPartner(partner);
}
}
/***********************************************
********** PRIVATE CLASS METHODS **********
***********************************************/
/**
* Create WIG file of predicted UTRs that
* can be loaded into a genome browser.
*/
private static void outputUTRsForBrowser(String outputFile, String genomeName, ArrayList<Gene> genes, int size) {
// Determine coordinates of UTRs
int[] geneCoordinates = new int[size];
for (int i=0; i<genes.size(); i++) {
Gene g = genes.get(i);
// UTRs
if (g.isORF() && (g.getStartT() > 0) && (g.getStrand() == '+')) // 5'UTR on plus strand
for (int j=g.getStartT(); j<g.getStart(); j++) geneCoordinates[j] = 1;
if (g.isORF() && (g.getStartT() > 0) && (g.getStrand() == '-')) // 5'UTR on minus strand
for (int j=g.getStart()+1; j<=g.getStartT(); j++) geneCoordinates[j] = -1;
if (g.isORF() && (g.getStopT() > 0) && (g.getStrand() == '+')) // 3'UTR on plus strand
for (int j=g.getStop()+1; j<=g.getStopT(); j++) geneCoordinates[j] = 1;
if (g.isORF() && (g.getStopT() > 0) && (g.getStrand() == '-')) // 3'UTR on minus strand
for (int j=g.getStopT(); j<g.getStop(); j++) geneCoordinates[j] = -1;
}
// Output differentially expressed genes to genome browser file
try {
PrintWriter writer = new PrintWriter(new File(outputFile));
writer.println("track name=" + "\"" + "UTRs" + "\"" + " color=255,0,255 altColor=255,0,255 graphType=bar viewLimits=-1:1");
writer.println("fixedStep chrom=" + genomeName + " start=1 step=1");
for (int j=1; j<geneCoordinates.length; j++) writer.println(geneCoordinates[j]);
writer.close();
} catch (FileNotFoundException e) {
output("\nError - could not open file " + outputFile + "\n\n");
}
}
/**
* Create WIG file of predicted RNAs that
* can be loaded into a genome browser.
*/
private static void outputRNAsForBrowser(String outputFile, String genomeName, ArrayList<Gene> genes, int size) {
// Determine coordinates of ncRNAs
int[] geneCoordinates = new int[size];
for (int i=0; i<genes.size(); i++) {
Gene g = genes.get(i);
// ncRNAs
if (!g.isORF() && (g.getName().equals("predicted RNA"))) { // novel RNA
int value = 1;
if (g.getStrand() == '-') value = -1;
for (int j=g.getFirst(); j<=g.getLast(); j++) geneCoordinates[j] = value;
}
}
// Output differentially expressed genes to genome browser file
try {
PrintWriter writer = new PrintWriter(new File(outputFile));
writer.println("track name=" + "\"" + "Novel RNAs" + "\"" + " color=0,255,0 altColor=0,255,0 graphType=bar viewLimits=-1:1");
writer.println("fixedStep chrom=" + genomeName + " start=1 step=1");
for (int j=1; j<geneCoordinates.length; j++) writer.println(geneCoordinates[j]);
writer.close();
} catch (FileNotFoundException e) {
output("\nError - could not open file " + outputFile + "\n\n");
}
}
/**
* Create WIG file of differentially expressed genes that
* can be loaded into a genome browser.
*/
private static void outputDifferentiallyExpressedGenesForBrowser(String outputFile, String genomeName, ArrayList<Gene> genes, int size) {
// Determine coordinates of differentially expressed genes
int[] geneCoordinates = new int[size];
for (int i=0; i<genes.size(); i++) {
Gene g = genes.get(i);
double qValue = g.getMinQvalue();
int value = 0;
if (qValue == 0.0) value = 300; // Special case. We cannot take log of zero.
else value = (int)(-Math.log10(qValue));
if (g.getStrand() == '-') value = -value;
for (int j=g.getFirst(); j<=g.getLast(); j++) geneCoordinates[j] = value;
}
// Output differentially expressed genes to genome browser file
try {
PrintWriter writer = new PrintWriter(new File(outputFile));
writer.println("track name=" + "\"" + "Differentially expressed genes" + "\"" + " color=0,255,0 altColor=0,255,0 graphType=bar viewLimits=-10:10");
writer.println("fixedStep chrom=" + genomeName + " start=1 step=1");
for (int j=1; j<geneCoordinates.length; j++) writer.println(geneCoordinates[j]);
writer.close();
} catch (FileNotFoundException e) {
output("\nError - could not open file " + outputFile + "\n\n");
}
}
/**
* Returns the system-dependent file separator that
* can be used as a RegEx.
*/
private static String separator() {
if (File.separatorChar == '\\') return "\\\\";
else return File.separator;
}
/**********************************************
********** PUBLIC CLASS METHODS **********
**********************************************/
// Used for updating the GUI progress bar.
public static void updateProgress() {
if (worker != null) {
double totalStages = 0.0;
for (String files : conditionFiles) totalStages += files.split(",").length;
if (isDeNovo) {
for (String files : conditionFiles) totalStages += files.split(",").length;
for (String files : conditionFiles) totalStages += files.split(",").length;
}
totalStages += 1.0; // For analyzing transcripts and computing expression
stagesProcessed++;
worker.firePropertyChange("progress", worker.getProgress(), (int)(100.0*stagesProcessed/totalStages));
}
}
public static void commandLineArguments(String[] args) {
if (args.length < 1) {
output("\n*************************************************\n");
output("********** Rockhopper version " + Rockhopper.version + " **********\n");
output("*************************************************\n");
output("\nThe Rockhopper application has the following required command line arguments.\n");
output("\nREQUIRED ARGUMENTS\n\n");
output("\t" + "exp1A.fastq,exp1B.fastq,exp1C.fastq exp2A.fastq,exp2B.fastq" + "\t" + "a comma separated list of sequencing files (in FASTQ, QSEQ, FASTA, SAM, or BAM format) for replicate experiments, one list per experimental condition (mate-pair files should be delimited by '%')\n");
output("\nREFERENCE BASED ASSEMBLY VS. DE NONO ASSEMBLY:\n");
output("IF THE -g OPTION IS USED THEN ROCKHOPPER ALIGNS READS TO ONE OR MORE REFERENCE GENOMES,\n");
output("OTHERWISE, ROCKHOPPER PERFORMS DE NOVO TRANSCRIPT ASSEMBLY.\n\n");
output("\t" + "-g <DIR1,DIR2>" + "\t" + "a comma separated list of directories, each containing a genome file (*.fna), gene file (*.ptt), and rna file (*.rnt)\n");
output("\nOPTIONAL ARGUMENTS FOR EITHER REFERENCE BASED ASSEMBLY OR DE NOVO ASSEMBLY\n\n");
output("\t" + "-c <boolean>" + "\t" + "reverse complement single-end reads (default is false)\n");
output("\t" + "-ff/fr/rf/rr" + "\t" + "orientation of two mate reads for paired-end read, f=forward and r=reverse_complement (default is fr)\n");
output("\t" + "-d <integer>" + "\t" + "maximum number of bases between mate pairs for paired-end reads (default is 500)\n");
output("\t" + "-a <boolean>" + "\t" + "identify 1 alignment (true) or identify all optimal alignments (false), (default is true)\n");
output("\t" + "-p <integer>" + "\t" + "number of processors (default is self-identification of processors)\n");
output("\t" + "-e <boolean>" + "\t" + "compute differential expression for transcripts in pairs of experimental conditions (default is true)\n");
output("\t" + "-s <boolean>" + "\t" + "RNA-seq experiments are strand specific (true) or strand ambiguous (false), (default is true)\n");
output("\t" + "-L <comma separated list>" + "\t" + "labels for each condition\n");
output("\t" + "-o <DIR>" + "\t" + "directory where output files are written (default is Rockhopper_Results/)\n");
output("\t" + "-v <boolean>" + "\t" + "verbose output including raw/normalized counts aligning to each gene (default is false)\n");
output("\t" + "-SAM " + "\t" + "output a SAM format file\n");
output("\t" + "-TIME " + "\t" + "output time taken to execute program\n");
output("\nOPTIONAL ARGUMENTS FOR REFERENCE BASED ASSEMBLY ONLY\n\n");
output("\t" + "-m <number>" + "\t" + "allowed mismatches as percent of read length (default is 0.15)\n");
output("\t" + "-l <number>" + "\t" + "minimum seed as percent of read length (default is 0.33)\n");
output("\t" + "-y <boolean>" + "\t" + "compute operons (default is true)\n");
output("\t" + "-t <boolean>" + "\t" + "identify transcript boundaries including UTRs and ncRNAs (default is true)\n");
output("\t" + "-z <number>" + "\t" + "minimum expression of UTRs and ncRNAs, a number in range [0.0, 1.0] (default is 0.5)\n");
output("\nOPTIONAL ARGUMENTS FOR DE NOVO ASSEMBLY ONLY\n\n");
output("\t" + "-k <integer>" + "\t" + "size of k-mer, range of values is 15 to 31 (default is 25)\n");
output("\t" + "-j <integer>" + "\t" + "minimum length required to use a sequencing read after trimming/processing (default is 35)\n");
output("\t" + "-n <integer>" + "\t" + "size of k-mer hashtable is ~ 2^n (default is 25). HINT: should normally be 25 or, if more memory is available, 26. WARNING: if increased above 25 then more than 1.2M of memory must be allocated\n");
output("\t" + "-b <integer>" + "\t" + "minimum number of full length reads required to map to a de novo assembled trancript (default is 20)\n");
output("\t" + "-u <integer>" + "\t" + "minimum length of de novo assembled transcripts (default is 2*k)\n");
output("\t" + "-w <integer>" + "\t" + "minimum count of k-mer to use it to seed a new de novo assembled transcript (default is 50)\n");
output("\t" + "-x <integer>" + "\t" + "minimum count of k-mer to use it to extend an existing de novo assembled transcript (default is 5)\n");
output("\nEXAMPLE EXECUTION: REFERENCE BASED ASSEMBLY WITH SINGLE-END READS\n");
output("\nrockhopper <options> -g genome_DIR1,genome_DIR2 aerobic_replicate1.fastq,aerobic_replicate2.fastq anaerobic_replicate1.fastq,anaerobic_replicate2.fastq\n");
output("\nEXAMPLE EXECUTION: REFERENCE BASED ASSEMBLY WITH PAIRED-END READS\n");
output("\nrockhopper <options> -g genome_DIR1,genome_DIR2 aerobic_replicate1_pairedend1.fastq%aerobic_replicate1_pairedend2.fastq,aerobic_replicate2_pairedend1.fastq%aerobic_replicate2_pairedend2.fastq anaerobic_replicate1_pairedend1.fastq%anaerobic_replicate1_pairedend2.fastq,anaerobic_replicate2_pairedend1.fastq%anaerobic_replicate2_pairedend2.fastq\n");
output("\nEXAMPLE EXECUTION: DE NOVO ASSEMBLY WITH SINGLE-END READS\n");
output("\nrockhopper <options> aerobic_replicate1.fastq,aerobic_replicate2.fastq anaerobic_replicate1.fastq,anaerobic_replicate2.fastq\n");
output("\nEXAMPLE EXECUTION: DE NOVO ASSEMBLY WITH PAIRED-END READS\n");
output("\nrockhopper <options> aerobic_replicate1_pairedend1.fastq%aerobic_replicate1_pairedend2.fastq,aerobic_replicate2_pairedend1.fastq%aerobic_replicate2_pairedend2.fastq anaerobic_replicate1_pairedend1.fastq%anaerobic_replicate1_pairedend2.fastq,anaerobic_replicate2_pairedend1.fastq%anaerobic_replicate2_pairedend2.fastq\n");
output("\n");
System.exit(0);
}
// Initially set the number of threads
Peregrine.numThreads = Runtime.getRuntime().availableProcessors();
if (Peregrine.numThreads > 4) Peregrine.numThreads *= 0.75;
Assembler.numThreads = Runtime.getRuntime().availableProcessors();
if (Assembler.numThreads > 4) Assembler.numThreads = (int)Math.min(Assembler.numThreads * 0.75, 8.0);
int i = 0;
conditionFiles = new ArrayList<String>();
genome_DIRs = null;
while (i < args.length) {
if (args[i].equals("-g")) {
if ((i == args.length-1) || (args[i+1].startsWith("-"))) {
output("Error - command line argument -g must be followed by the name of one or more directories.\n");
System.exit(0);
}
String[] parse_dirs = args[i+1].split(",");
genome_DIRs = new ArrayList<String>();
for (String s : parse_dirs) {
if (s.endsWith(File.separator)) genome_DIRs.add(s);
else genome_DIRs.add(s + File.separatorChar);
}
isDeNovo = false;
i += 2;
} else if (args[i].startsWith("-a")) {
if ((i == args.length-1) || (args[i+1].startsWith("-"))) {
output("Error - command line argument -a must be followed by a boolean.\n");
System.exit(0);
}
Peregrine.stopAfterOneHit = Boolean.valueOf(args[i+1]);
Assembler.stopAfterOneHit = Boolean.valueOf(args[i+1]);
i += 2;
} else if (args[i].startsWith("-p")) {
if ((i == args.length-1) || (args[i+1].startsWith("-"))) {
output("Error - command line argument -p must be followed by an integer.\n");
System.exit(0);
}
Peregrine.numThreads = Integer.parseInt(args[i+1]);
Assembler.numThreads = Integer.parseInt(args[i+1]);
i += 2;
} else if (args[i].startsWith("-e")) {
if ((i == args.length-1) || (args[i+1].startsWith("-"))) {
output("Error - command line argument -e must be followed by a boolean.\n");
System.exit(0);
}
computeExpression = Boolean.valueOf(args[i+1]);
Assembler.computeExpression = Boolean.valueOf(args[i+1]);
i += 2;
} else if (args[i].startsWith("-s")) {
if ((i == args.length-1) || (args[i+1].startsWith("-"))) {
output("Error - command line argument -s must be followed by a boolean.\n");
System.exit(0);
}
unstranded = !Boolean.valueOf(args[i+1]);
Assembler.unstranded = !Boolean.valueOf(args[i+1]);
i += 2;
} else if (args[i].startsWith("-d")) {
if ((i == args.length-1) || (args[i+1].startsWith("-"))) {
output("Error - command line argument -d must be followed by an integer.\n");
System.exit(0);
}
Peregrine.maxPairedEndLength = Integer.parseInt(args[i+1]);
Assembler.maxPairedEndLength = Integer.parseInt(args[i+1]);
SamOps.maxPairedEndLength = Integer.parseInt(args[i+1]);
i += 2;
} else if (args[i].equals("-L")) {
if (i == args.length-1) {
output("Error - command line argument -L must be followed by a comma separated list of names for the conditions.\n");
System.exit(0);
}
labels = args[i+1].split(",");
Assembler.labels = args[i+1].split(",");
i += 2;
} else if (args[i].equals("-o")) {
if ((i == args.length-1) || (args[i+1].startsWith("-"))) {
output("Error - command line argument -o must be followed the name of a directory.\n");
System.exit(0);
}
if (args[i+1].endsWith("/")) output_DIR = args[i+1];
else output_DIR = args[i+1] + "/";
Peregrine.outputDIR = output_DIR;
Assembler.output_DIR = output_DIR;
i += 2;
} else if (args[i].startsWith("-v")) {
if ((i == args.length-1) || (args[i+1].startsWith("-"))) {
output("Error - command line argument -v must be followed by a boolean.\n");
System.exit(0);
}
verbose = Boolean.valueOf(args[i+1]);
Assembler.verbose = Boolean.valueOf(args[i+1]);
i += 2;
} else if (args[i].startsWith("-m")) {
if ((i == args.length-1) || (args[i+1].startsWith("-"))) {
output("Error - command line argument -m must be followed by a decimal number.\n");
System.exit(0);
}
Peregrine.percentMismatches = Double.parseDouble(args[i+1]);
i += 2;
} else if (args[i].startsWith("-l")) {
if ((i == args.length-1) || (args[i+1].startsWith("-"))) {
output("Error - command line argument -l must be followed by a decimal number.\n");
System.exit(0);
}
Peregrine.percentSeedLength = Double.parseDouble(args[i+1]);
i += 2;
} else if (args[i].startsWith("-y")) {
if ((i == args.length-1) || (args[i+1].startsWith("-"))) {
output("Error - command line argument -y must be followed by a boolean.\n");
System.exit(0);
}
computeOperons = Boolean.valueOf(args[i+1]);
i += 2;
} else if (args[i].startsWith("-t")) {
if ((i == args.length-1) || (args[i+1].startsWith("-"))) {
output("Error - command line argument -t must be followed by a boolean.\n");
System.exit(0);
}
computeTranscripts = Boolean.valueOf(args[i+1]);
i += 2;
} else if (args[i].equals("-z")) {
if ((i == args.length-1) || (args[i+1].startsWith("-"))) {
output("Error - command line argument -z must be followed by a number in the range [0.0,1.0].\n");
System.exit(0);
}
transcriptSensitivity = Double.parseDouble(args[i+1]);
i += 2;
} else if (args[i].startsWith("-k")) {
if ((i == args.length-2) || (args[i+1].startsWith("-"))) {
output("Error - command line argument -k must be followed by an integer.\n");
System.exit(0);
}
Assembler.k = Integer.parseInt(args[i+1]);
if (Assembler.minTranscriptLength == 0) Assembler.minTranscriptLength = 2*Assembler.k;
i += 2;
} else if (args[i].startsWith("-j")) {
if ((i == args.length-2) || (args[i+1].startsWith("-"))) {
output("Error - command line argument -j must be followed by an integer.\n");
System.exit(0);
}
Assembler.minReadLength = Integer.parseInt(args[i+1]);
i += 2;
} else if (args[i].startsWith("-n")) {
if ((i == args.length-2) || (args[i+1].startsWith("-"))) {
output("Error - command line argument -n must be followed by an integer.\n");
System.exit(0);
}
Assembler.CAPACITY_POWER = Integer.parseInt(args[i+1]);
i += 2;
} else if (args[i].startsWith("-b")) {
if ((i == args.length-2) || (args[i+1].startsWith("-"))) {
output("Error - command line argument -b must be followed by an integer.\n");
System.exit(0);
}
Assembler.MIN_READS_MAPPING = Integer.parseInt(args[i+1]);
i += 2;
} else if (args[i].startsWith("-u")) {
if ((i == args.length-2) || (args[i+1].startsWith("-"))) {
output("Error - command line argument -u must be followed by an integer.\n");
System.exit(0);
}
Assembler.minTranscriptLength = Integer.parseInt(args[i+1]);
i += 2;
} else if (args[i].startsWith("-w")) {
if ((i == args.length-2) || (args[i+1].startsWith("-"))) {
output("Error - command line argument -w must be followed by an integer.\n");
System.exit(0);
}
Assembler.minSeedExpression = Integer.parseInt(args[i+1]);
i += 2;
} else if (args[i].startsWith("-x")) {
if ((i == args.length-2) || (args[i+1].startsWith("-"))) {
output("Error - command line argument -x must be followed by an integer.\n");
System.exit(0);
}
Assembler.minExpression = Integer.parseInt(args[i+1]);
i += 2;
} else if (args[i].startsWith("-c")) {
if ((i == args.length-1) || (args[i+1].startsWith("-"))) {
output("Error - command line argument -c must be followed by a boolean.\n");
System.exit(0);
}
Peregrine.singleEndOrientationReverseComplement = Boolean.valueOf(args[i+1]);
Assembler.singleEndOrientationReverseComplement = Boolean.valueOf(args[i+1]);
i += 2;
} else if (args[i].equals("-ff")) {
Peregrine.pairedEndOrientation = "ff";
Assembler.pairedEndOrientation = "ff";
i++;
} else if (args[i].equals("-fr")) {
Peregrine.pairedEndOrientation = "fr";
Assembler.pairedEndOrientation = "fr";
i++;
} else if (args[i].equals("-rf")) {
Peregrine.pairedEndOrientation = "rf";
Assembler.pairedEndOrientation = "rf";
i++;
} else if (args[i].equals("-rr")) {
Peregrine.pairedEndOrientation = "rr";
Assembler.pairedEndOrientation = "rr";
i++;
} else if (args[i].equals("-TIME")) {
time = true;
Assembler.time = true;
i++;
} else if (args[i].equals("-SAM")) {
Peregrine.outputSAM = true;
Assembler.outputSAM = true;
i++;
} else {
conditionFiles.add(args[i]);
i++;
}
}
// Handle erroneous command line arguments
if (genome_DIRs == null) {
isDeNovo = true;
} else {
for (int j=0; j<genome_DIRs.size(); j++) {
if (!(new File(genome_DIRs.get(j)).exists())) {
output("Error - directory " + genome_DIRs.get(j) + " does not exist.\n");
System.exit(0);
}
}
}
if (conditionFiles.size() == 0) {
output("Error - sequencing reads files (in FASTQ, QSEQ, FASTA, SAM, or BAM format) are required as command line arguments.\n");
System.exit(0);
}
Assembler.conditionFiles = conditionFiles;
Assembler.expressionFile = expressionFile;
Assembler.output_DIR = output_DIR;
Peregrine.outputDIR = output_DIR;
for (int j=0; j<conditionFiles.size(); j++) {
String[] files = conditionFiles.get(j).split(",");
for (int k=0; k<files.length; k++) {
String[] pairedEnd_files = files[k].split("%");
if (!(new File(pairedEnd_files[0]).exists())) {
output("Error - file " + pairedEnd_files[0] + " does not exist.\n");
System.exit(0);
}
if ((pairedEnd_files.length > 1) && (!(new File(pairedEnd_files[1]).exists()))) {
output("Error - file " + pairedEnd_files[1] + " does not exist.\n");
System.exit(0);
}
if (pairedEnd_files.length > 1) Assembler.unstranded = false; // Assume paired-end reads are strand specific
}
}
// If not set by command line arguments, set de novo assembled transcript length
if (Assembler.minTranscriptLength == 0) Assembler.minTranscriptLength = 2*Assembler.k;
// Output directory does not exist. Create it.
if (!(new File(output_DIR).exists())) {
if (!(new File(output_DIR).mkdir())) {
output("Error - could not create directory " + output_DIR + "." + "\n");
System.exit(0);
}
}
Peregrine.browserDIR = Rockhopper.browser_DIR;
if (!(new File(output_DIR + browser_DIR).exists())) {
if (!(new File(output_DIR + browser_DIR).mkdir())) {
output("Error - could not create directory " + (output_DIR+browser_DIR) + "." + "\n");
System.exit(0);
}
}
try {
summaryWriter = new PrintWriter(new File(output_DIR + summaryFile));
Peregrine.summaryWriter = summaryWriter;
Assembler.summaryWriter = summaryWriter;
} catch (FileNotFoundException e) {
output("Error - could not create file " + (output_DIR+summaryFile) + "\n");
System.exit(0);
}
}
public static void output(String s) {
if (summaryWriter != null) summaryWriter.print(s);
if (output == null) System.out.print(s);
else {
output.append(s);
output.setCaretPosition(output.getDocument().getLength());
}
}
/*************************************
********** MAIN METHOD **********
*************************************/
/**
* The Main method, when invoked with the appropriate command line
* arguments, executes the Rockhopper application for the purpose
* of analyzing RNA-seq data.
*/
public static void main(String[] args) {
commandLineArguments(args);
long runningTime = System.currentTimeMillis();
Rockhopper rh = new Rockhopper();
runningTime = System.currentTimeMillis() - runningTime;
if (time) { // Output time taken to execute program
System.out.println("Execution time:\t" + (runningTime/60000) + " minutes " + ((runningTime%60000)/1000) + " seconds" + "\n");
}
}
}
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