scripture with paired-end data - problem

[NicoBxl]

Hi,

I've problem with scripture.

I performed alignments with tophat 2 (paired-end, 76bp) and now want to use scripture to build a transcriptome. When I check the scripture help, I don't understand how to use it.

Here's the help :

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>

My questions :

The accepted_hits.bam from tophat, should I input it with -alignment or -pairedEnd.
What is -maskFileDir and -windows param ?


Thanks a lot,

N.

[rboettcher]

Hi Nico,

since both cufflinks and scriptute are provided by the Broad institute, my guess is that both have similar input parameters. That in mind, the maskFileDir should contain a gtf file with known regions that should be excluded ("masked") from the assembly process to focus on novel transcripts only.

Looking at the walktrough provided on the Scripture website
(http://www.broadinstitute.org/softwa...hrough_example),
they suggest to concatenate your paired end alignment files and then use the -alignment flag.

I'm interested in the outcome of your analysis in general (especially compared to cufflinks), so please keep me posted.

Best regards,
René

[NicoBxl]

Thanks.

what about the -windows param ?

[rboettcher]

To anyone coming across this thread because they can't figure out how to set Scripture's -windows flag (Which is listed as optional in the documentation, but seems mandatory when running scripture.jar):

this problem can also be solved by using the latest beta-2 build, which is available from http://www.broadinstitute.org/softwa...re/?q=download

hope this helps

[bruce01]

I have been trying to run Scripture using Samtools merged .bam files for my conditions. The original .bam files are from Tophat2. When I run Scripture I get "Has pairs: false" when data is from paired-end sequence. Do you have to give the original .fq files? What comes after -pairedData flag? In short: how do I get "Has pairs: true"?

Help much appreciated.

[rboettcher]

So I also tried scripture in beta version 2, after alignment with tophat2 (as recommended on the homepage), however I keep getting the following warning message:

[SamAlignment.java:243] [main] Warning: invalid quality string length. Read length = 101 quality string length = 0

The resulting files contain quite a lot of transcript estimates, still they all score 0.0.

Any suggestions?

[baibaijingjing]

I have got the same problem when I used the example data downloaded from ftp://ftp.ncbi.nih.gov/pub/geo/DATA/...aligned.sam.gz. but Has pairs is false and all the scores are 0. I hope someone would solve it .