SOPs/coordinates – BaRC Wiki

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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 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:
       <chromName><TAB><chromSize>
       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:
        <chromName><TAB><chromSize>

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 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

convert gtf to genePhred
```
   gtfToGenePred my.gtf my.genePhred
```

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

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"

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"

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"

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 RSeQC or visit BaRC SOP: SAM/BAM summarizing, processing and quality control(QC)
```
infer_experiment.py -r mm9.refseq.bed12 -i accepted_hits.bam
```

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

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