Scripture : Paired-End Data

Question

[Nicolas Rosewick]

Hi,

I've a little problem to understand the scripture command line :

So I've the accepted_hits.bam for all my samples. So I have to merge all the bam files into one big bam file before running scripture. In the help file there is two alignment file param :

-alignment --pairedEnd

Which one do I have to use to input the merged bam file ?

Here's the help file :

Parameters 
 -alignment <Alignment file in BAM, SAM or Alignemnt format> 
 -maskFileDir <Mask File directory> 
 -out <Output file name>
 -windows <Comma separated list of windows to evaluate>
 -sizeFile <Chromosome size file>  
 Optional arguments: 
 -trim <Include this flag if trimming of the ends of windows based on read coverage  is desired this is expensive> 
 -alpha <Desired FDR>
 one of -chr <Chromsomosome to segment> or -chrSequence <Necessary to filter spliced reads by splice site information. Notice that this is only compatible with region files that contain regions of only one chromosome> -dontFilterCanonicalSplice
 -start <To segment only a subregion of the chromosome include its start> -end <To segment only a subregion of the chromosome include its end>
 -minSpliceSupport <Minimum count to support splice reads, default is 1> 
 -pairedEnd <Paired end alignment files> -strandSpecificReads <Strand specific alignment file> -scoreRegions <Full BED to score> -upWeightSplices -lambda <If a prior background expectation for number of reads per base exists> -exons <BED file of exons> -introns <Introns and counts>

Task: AddPairs -  Uses a paired end alignment to tune graph 
    -in <Graph in .dot format. Standard input is assumed> 
    -pairedEnd <Paired end information (as in previous task), in single line BED format>
     -maskFileDir <Directory containing mask files for the genome> 
    -chr <Chromosome (only a chromosome at a time is supported at this point)> 
    -sizeFile <Chromosome size file> 
    -out <Output file name>

Task: score -  Computes several expression related scores for a set of annotations -in <Full BED file with annotations to score> 
    -alignment <Alignment file in BAM, SAM or Alignemnt format> 
    -sizeFile <Chromosome size file> 
    -out <Output file name> 
     -maskFileDir <Mask File directory>

Thanks,

N.

Answer 1

[Vikas Bansal]

I just read about this tool and will share what I understood. So you can read a walkthrough example here.So if you have paired end data, then you have option to use pairedEnd argument. This argument will take files generated by makePairedFile task.

From the link- So if you have paired end data, you will have 2 files for each "accepted_hits.sorted.sam". First you have to use makePairedFile task-

>> java -Xmx4000m -jar scripture.jar -task makePairedFile -pair1 tophat_out_SRR039999_1/accepted_hits.sorted.sam -pair2 tophat_out_SRR039999_2/accepted_hits.sorted.sam -out SRR039999.paired.sam -sorted
>> java -Xmx4000m -jar scripture.jar -task makePairedFile -pair1 tophat_out_SRR040000_1/accepted_hits.sorted.sam -pair2 tophat_out_SRR040000_2/accepted_hits.sorted.sam -out SRR040000.paired.sam -sorted
>> java -Xmx4000m -jar scripture.jar -task makePairedFile -pair1 tophat_out_SRR040001_1/accepted_hits.sorted.sam -pair2 tophat_out_SRR040001_2/accepted_hits.sorted.sam -out SRR040001.paired.sam -sorted

and then combine these files-

>> cat SRR039999.paired.sam SRR040000.paired.sam SRR040001.paired.sam > all_alignments.paired.sam

and then sort and index. So now this file "all_alignments.paired.sorted.sam" will be used in pairedEnd argument. and for -alignment parameter you will simple combine your bam files-

>> cat tophat_out_SRR039999_1/accepted_hits.sorted.sam tophat_out_SRR039999_2/accepted_hits.sorted.sam tophat_out_SRR040000_1/accepted_hits.sorted.sam tophat_out_SRR040000_2/accepted_hits.sorted.sam tophat_out_SRR040001_1/accepted_hits.sorted.sam tophat_out_SRR040001_2/accepted_hits.sorted.sam > all_alignments.sam

Sort and index.Now this "all_alignments.sorted.sam" will be used in -alignment parameter.

So they used this command in example-

>>java –jar scripture.jar –alignment all_alignments.sorted.sam –out chr19.scriptureESTest.segments –sizeFile mm9.sizes –chr chr19 –chrSequence chr19.fa -pairedEnd all_alignments.paired.sorted.sam