Scripture - transcriptome reconstruction

[Boel]

This paper was published last week, and introduces a new transcriptome reconstruction program: Scripture.

Ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs.

Guttman M, Garber M, Levin JZ, Donaghey J, Robinson J, Adiconis X, Fan L, Koziol MJ, Gnirke A, Nusbaum C, Rinn JL, Lander ES, Regev A.

Nat Biotechnol. 2010 May 2. [Epub ahead of print]
PMID: 20436462

Scripture is available here:

http://www.broadinstitute.org/software/scripture

[Wei-HD]

It is applicable for pair end reads, what about single end?

[Thomas Doktor]

The example walk-through seems to use a non-paired SAM alignment file, so my guess is yes, it supports single end reads.

[choy]

Does Scripture support SAM files with multiple biological replicates?

Hi,

I am interested in trying out the Scripture tool.

I have three samples that are part of a time course. Each sample was aligned by TopHat and has an associated SAM file.

Can I aggregate all three SAM files together into one before running Scripture? Does Scripture assume a single insert size distribution for the entire SAM file?

In one of my samples, I trimmed the sequences at the 3' end to improve alignment (QC suggested high error rate at the 3' end of the reads in that sample). Can Scripture handle different read lengths (a single SAM file with a mix of read lengths) in this case?

What if the read lengths varied during sequencing? Can I mix together 2x40bp and 2x75bp reads into a single SAM file for Scripture?

Finally, how does Scripture handle multireads? I know that TopHat has a setting for the maximum multiread hits tolerated per sequence. How does Scripture make use of this information? Do you recommend we run TopHat and keep only the uniquely mapping reads?

Thanks for your help!

[NextGenSeq]

What a terrible name. It sounds like a faith based bible chapter than a logical scientific based tool. Did LiCor develop it?

[choy]

Scripture help

Anyone have any insight regarding my questions above? Has anyone else tried Scripture?

[Boel]

Hi choy,

I have tried Scripture on my data.
Merging all the SAM files before the first step is a good idea. I do not know whether a single insert size is assumed, or how multi-mapping reads are handled.

In order to assemble the transcriptome paired end data is pretty crucial, but I do not think that it is impossible to run the tool on single end.

Sorry that I am not able to give any more detailed answers! This tool is still in its infancy, but does assemble full length transcripts - something that Cufflinks has some problems doing.

[Wei-HD]

Hi Boel,

Can you explain to me why it is better to concatenate all the TopHat output sam file together? I do not get it here because the sam files are from different biological samples.

Many thanks!
Wei

[Boel]

In the first step (segmentation) you are creating the graph so there it is better to use all the reads you have to get more power in detecting all junctions and present exons. In later steps where you want to get information on the transcripts expressed in one particular sample you would only use the bam (sam) file from that sample.

[Wei-HD]

Hi Boel,

Thanks a lot!

So that means I combined all the sam files from different samples together and get the whole all_alignments.sam file. For example: I have conditionA.sam file and conditionB.sam file. Afterwards, I run Scripture with each chromosome 1, and the output files include bed file and dot file, is this the segmentation step?

Sorry my head got stuck here, but what you mean the later steps. And the dot file and bed file are from the all_alignments.sam file, how to differentiate different samples.

Many thanks!

[adeslat]

scripture bug ?

I have run scripture on my ABI solid paired end read data set after preparing it as instructed.

When running scripture I get an error unfortunately no output.

Note that the hg19_c.summary.txt file has the following as its content:
I made this file from running the following bowtie-inspect command on my hg19_c (color index file for hg release 19):
bowtie-inspect -s hg19_c

chr1 249250621
chr2 243199373
chr3 198022430
chr4 191154276
chr5 180915260
chr6 171115067
chr7 159138663
chr8 146364022
chr9 141213431
chr10 135534747
chr11 135006516
chr12 133851895
chr13 115169878
chr14 107349540
chr15 102531392
chr16 90354753
chr17 81195210
chr18 78077248
chr19 59128983
chr20 63025520
chr21 48129895
chr22 51304566
chrX 155270560
chrY 59373566
chrM 16571

I'm running scripture with the following command:

java -jar scripture.jar -alignment sorted.bam -out chr2.scriptureEST.segments -windows 200\
-sizeFile hg19_c.summary.txt -chr chr2 -chrSequence chr2.fa -pairedEnd bmdeplet.sorted.bam

Thoughts?

Here is the error:

Finished 2nd Graph construction
Exception in thread "main" java.lang.IllegalStateException: Iteration in progress
at net.sf.samtools.BAMFileReader.query(BAMFileReader.java:177)
at net.sf.samtools.SAMFileReader.query(SAMFileReader.java:221)
at org.broad.igv.sam.reader.BAMQueryReader.query(BAMQueryReader.java:74)
at broad.pda.seq.segmentation.GenericAlignmentDataModel.getReadIterator(GenericAlignmentDataModel.java:72)
at broad.pda.seq.segmentation.AlignmentDataModelStats.countWithinExon(AlignmentDataModelStats.java:316)
at broad.pda.seq.segmentation.ContinuousDataAlignmentModel.countExons(ContinuousDataAlignmentModel.java:1348)
at broad.pda.seq.segmentation.ContinuousDataAlignmentModel.makeGraphs(ContinuousDataAlignmentModel.java:1333)
at broad.pda.seq.segmentation.ContinuousDataAlignmentModel.acrossGraph(ContinuousDataAlignmentModel.java:770)
at broad.pda.seq.segmentation.ContinuousDataAlignmentModel.scanGenome(ContinuousDataAlignmentModel.java:484)
at broad.pda.seq.segmentation.ContinuousDataAlignmentModel.scanFromGraph(ContinuousDataAlignmentModel.java:373)
at broad.pda.seq.segmentation.ContinuousDataAlignmentModel.main(ContinuousDataAlignmentModel.java:2064)

[adeslat]

scripture-beta.jar

Hi all,

The bug was overcome by running scripture-beta.jar file instead.

Problem solved!

Anne

[ozs2006]

hi,
I'm interested using scripture for RNA-seq analysis of alternative splicing.
Is there a detailed documentation for all the program's flags, (I'm having some trouble making the example at scripture website works on my computer)?

[Eric Fournier]

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

[nbartonicek]

I'm trying to asses the reads through scripture (using Cufflinks, and wanted to have a point of comparison).

For some reason, it calls on the X server, though there is no graphical display (?) and complains though my X11 functions normally.

Exception in thread "main" java.lang.InternalError: Can't connect to X11 window server using 'localhost:27.0' as the value of the DISPLAY variable.

Is there a way to disable display forwarding?

Cheers,

Nenad