* The detailed format descriptions can be found at http://genome.ucsc.edu/goldenPath/help/customTrack.html * How to choose data format? Check http://genomewiki.ucsc.edu/index.php/Selecting_a_graphing_track_data_format * samtools reference: http://samtools.sourceforge.net/ * samtools and bedtools have been installed on tak * Many other applications are available from [https://hgdownload.cse.ucsc.edu/admin/exe/ UCSC Bioinformatics]. * [Whitehead only] Other scripts can be found in the BaRC_Public/BaRC_code folder (/nfs/BaRC_Public/BaRC_code) === Convert bed to wig === {{{ Sample command: bed2wig.pl inputBed sampleName(one word) probeWidth > outputWig Note: It assumes that the probe width in all records is constant. If probe width is not constant, you can use bedGraph format. To convert bed to bedGraph format, just change the track name to bedGraph, and minus chromosome end position in bed format by 1. }}} === Convert wig to bed === {{{ Sample command with variableStep wig format: wig2bed.pl inputWig sampleName(one word) > outputBed Sample command with fixedStep wig format: wig2bed_fixedStep.pl inputWig > outputBed }}} === Convert wig to bigwig === {{{ Sample commands: Get chromosome lengths fetchChromSizes hg18 > chrSize.txt Convert wig to big wig: wigToBigWig foo.wig chrSize.txt foo.bw }}} === Convert bed to bigbed === {{{ Sample commands: Get chromosome lengths fetchChromSizes hg18 > chrSize.txt Convert bed to big bed: bedToBigBed foo.bed chrSize.txt foo.bb }}} === Convert BAM to bedGraph for UCSC genome browser === {{{ To view BAM files on UCSC browser, both foo.sorted.bam and foo.sorted.bam.bai have to be on a http or ftp server. One way to get around this is to convert BAM files into bedGraph files, which should be small enough that they can be simply uploaded. genomeCoverageBed -split -bg -ibam sorted.bam -g hg19.genome where hg19.genome file is tab delimited and structured as follows: chr1 249250621 One can use the UCSC Genome Browser's MySQL database to extract chromosome sizes. For example, H. sapiens: mysql --user=genome --host=genome-mysql.cse.ucsc.edu -A -e "select chrom, size from hg19.chromInfo" > hg19.genome }}} === Convert bam to bigwig === Method 1: Single-base resolution across the genome {{{ Step1: convert bam to bedGraph format: genomeCoverageBed -split -bg -ibam accepted_hits.bam -g /nfs/genomes/mouse_gp_jul_07/anno/mm9.size > accepted_hits.bedGraph Step2: convert bedGraph to bigwig format: bedGraphToBigWig accepted_hits.bedGraph /nfs/genomes/mouse_gp_jul_07/anno/mm9.size accepted_hits.bw where mm9.size file is tab delimited and structured as follows: }}} Method 2: Resolution of desired window size (after creating windows across desired regions or genome) {{{ coverageBed -a Human.hg19.1000.500.bed -b Sample_1.sorted.bam | cut -f1-4 > Sample_1.1000.500.coverage.bedgraph }}} === Update/fix UCSC GTF file === * GTF files from UCSC Table Browser use RefSeq (NM* ids) for both gene_id and transcript_id which may not be compatible for some programs (eg. counting by genes using HTSeq) * Some Refseq gtf files (such as for the hg19, hg18, mm9, and dm3 assemblies) are in /nfs/genomes/, under gtf/ in each species folder. If you would like to create additional files, here are the steps: {{{ Step 1: Use UCSC Table Browser to download RefSeq id and gene symbol. Use "Genes and Gene Prediction Tracks" for group, "RefSeq Genes" for track and "refGene" for table. Choose "selected fields from primary and related tables" for output format and click "get output". In the next page select "name" and "name2" for the fields. output format should be : NM_017940 NBPF1 Step 2: Download a gtf file from the UCSC Table Browser This uses refseq ID as gene_id and transcript_id, so we need to replace it with the gene symbol. sample command: /nfs/BaRC_Public/BaRC_code/Perl/fix_gtf_refSeq_ensembl.pl hg19.refgene.gtf refseq2symbol > hg19.refgene.gtf Step 3: About 50-70 genes in the gtf file from UCSC are incorrect; they include exons with a start coordinate that is larger than the end coordinate. Software such as cufflinks fails to deal with this situation and ignores these exons. Since this only affects the last 1-3 bases of a transcript, a temporary solution is to remove these records. sample command: awk -F"\t" '{ if($4<=$5) print $0 }' hg19.refgene.gtf > hg19.refgene_new.gtf }}} * Ensembl gtf files can be downloaded from ftp://ftp.ensembl.org/pub/current_gtf/ === Convert bed to gff === * Note that bed and gff use slightly different coordinate conventions * Use /nfs/BaRC_Public/BaRC_code/Perl/bed2gff/bed2gff.pl {{{ USAGE: bed2gff.pl bedFile > gffFile Ex: bed2gff.pl foo.bed WIBR exon > foo.gff }}} === Split bed file by chromosome === * Sometimes it's easier working with only one chromosome of regions at a time * Output files will be named like "Sample_1.chr1.bed". {{{ awk '{close(f);f=$1}{print > "Sample_1."f".bed"}' Sample_1_all_chrs.bed }}} === Convert gff to gtf === Use [http://ccb.jhu.edu/software/stringtie/gff.shtml gffread]: Try 'gffread -h' too see the program's many options {{{ gffread My_transcripts_genes.gff3 -T -E -o My_transcripts_genes.gtf }}} === Convert gtf to bed === 1. convert gtf to genePhred {{{ gtfToGenePred my.gtf my.genePhred }}} 2. convert genePhred to bed: {{{ awk -f genePhredToBed my.genePhred > my.bed }}} genePhredToBed is a awk script by Katrina Learned, downloaded from UCSC Genome Browser discussion list {{{ #!/usr/bin/awk -f # # Convert genePred file to a bed file (on stdout) # BEGIN { FS="\t"; OFS="\t"; } { name=$1 chrom=$2 strand=$3 start=$4 end=$5 cdsStart=$6 cdsEnd=$7 blkCnt=$8 delete starts split($9, starts, ","); delete ends split($10, ends, ","); blkStarts="" blkSizes="" for (i = 1; i <= blkCnt; i++) { blkSizes = blkSizes (ends[i]-starts[i]) ","; blkStarts = blkStarts (starts[i]-start) ","; } print chrom, start, end, name, 1000, strand, cdsStart, cdsEnd, 0, blkCnt, blkSizes, blkStarts } }}} === Convert blat to gff === * Use /nfs/BaRC_Public/BaRC_code/Perl/blat2gff/blat2gff.pl {{{ Convert BLAT output file (PSL format) into GFF format (v1.1 14 Dec 2010) blat2gff.pl blatFile dataSource(ex:WIBR) > gffFile }}} === Create wiggle files for visualizing paired-end data mapping to the + and - strands === 1. split by strand by matched strand {{{ # input: accepted_hits.bam # output: accepted_hits_negStrand.bam: mapped to negative strand # accepted_hits_posStrand.bam: mapped to positive strand bsub "samtools view -f 16 -b accepted_hits.bam >| accepted_hits_negStrand.bam" bsub "samtools view -F 16 -b accepted_hits.bam >| accepted_hits_posStrand.bam" }}} 2. split reads by pair {{{ # input: accepted_hits_posStrand.bam or accepted_hits_negStrand.bam # output: 1st pair: *_1stPair.bam # 2nd pair: *_2ndPair.bam bsub "samtools view -b -f 0x0040 accepted_hits_posStrand.bam > accepted_hits_posStrand_1stPair.bam" bsub "samtools view -b -F 0x0040 accepted_hits_posStrand.bam > accepted_hits_posStrand_2ndPair.bam" bsub "samtools view -b -f 0x0040 accepted_hits_negStrand.bam > accepted_hits_negStrand_1stPair.bam" bsub "samtools view -b -F 0x0040 accepted_hits_negStrand.bam > accepted_hits_negStrand_2ndPair.bam" }}} 3. convert from bam to bedgraph format {{{ # input: bam format: accepted_hits_*Strand_*Pair.bam # /nfs/genomes/mouse_gp_jul_07/anno/mm9.size: length of each chromosome, format like # chr1 197195432 # output: bedgraph format: accepted_hits_*Strand_*Pair.bedgraph bsub "genomeCoverageBed -split -bg -ibam accepted_hits_posStrand_1stPair.bam -g mm9.size >| accepted_hits_posStrand_1stPair.bedgraph" bsub "genomeCoverageBed -split -bg -ibam accepted_hits_posStrand_2ndPair.bam -g mm9.size >| accepted_hits_posStrand_2ndPair.bedgraph" bsub "genomeCoverageBed -split -bg -ibam accepted_hits_negStrand_1stPair.bam -g mm9.size >| accepted_hits_negStrand_1stPair.bedgraph" bsub "genomeCoverageBed -split -bg -ibam accepted_hits_negStrand_2ndPair.bam -g mm9.size >| accepted_hits_negStrand_2ndPair.bedgraph" }}} 4. join the reads sharing the same strand {{{ # This step is for fr-firststrand library (such as dUTP). which is 1+-,1-+,2++,2-- read1 mapped to ‘+’ strand indicates parental gene on ‘-‘ strand read1 mapped to ‘-‘ strand indicates parental gene on ‘+’ strand read2 mapped to ‘+’ strand indicates parental gene on ‘+’ strand read2 mapped to ‘-‘ strand indicates parental gene on ‘-‘ strand # input: bedgraph file from the same strand # output: merged bedgraph: pos.bedgraph or neg.bedgraph unionBedGraphs -i accepted_hits_posStrand_2ndPair.bedgraph accepted_hits_negStrand_1stPair.bedgraph |awk '{ print $1"\t"$2"\t"$3"\t"$4+$5 }' >|pos.bedgraph unionBedGraphs -i accepted_hits_posStrand_1stPair.bedgraph accepted_hits_negStrand_2ndPair.bedgraph |awk '{ print $1"\t"$2"\t"$3"\t-"$4+$5 }' >|neg.bedgraph }}} * To check how reads were stranded for strand-specific RNA-seq data, you can run infer_experiment.py from [http://rseqc.sourceforge.net/ RSeQC] or visit [http://barcwiki.wi.mit.edu/wiki/SOPs/miningSAMBAM BaRC SOP: SAM/BAM summarizing, processing and quality control(QC)] {{{ infer_experiment.py -r mm9.refseq.bed12 -i accepted_hits.bam }}} 5. convert bedgraph to bigwig {{{ # get rid of header lines of mm9.size: the header line with "chrom size" is removed # input: mm9.size: length of each chromosome # output: mm9.size_noHeader tail --line=+2 mm9.size > mm9.size_noHeader # convert bedgraph to bigwig # input: bedgraph file: neg.bedgraph or pos.bedgraph # mm9.size_noHeader: length of each chromosome # *output: bigwig format: neg.bw or pos.bw # neg.bw or pos.bw can be visualized with IGV/UCSC genome browser bsub bedGraphToBigWig neg.bedgraph mm9.size_noHeader neg.bw bsub bedGraphToBigWig pos.bedgraph mm9.size_noHeader pos.bw }}}