}

}
}
if(count==269)
{
//File f1=new File("op");
System.out.println(""+x+""+y);
}
}
this is my program and when i run this i got Array out of bound exception in line if((b[i][j]==c[m][n])&&(c[m][n]==1))
pls could any one point out what is wrong over here.

the tarball contains is a file named README.txt which aims at getting started with handling the program. To get a list and descriptions for all available parameters, use

flux -t simulator —printParameters

or

flux -t simulator -o

Best

seems like you are using some invalid or outdated parameters for v1.0-RC5. As described in the file README.txt contained in the tarball, to get a list and descriptions for all available parameters of the Flux Simulator, use

flux -t simulator —printParameters

or alternatively

flux -t simulator -o

In brief, the parameter name to tell the program to produce FASTA/FASTQ files is "FASTA", quality values (i.e., a FASTQ file) are produced if a sequencing error model is provided. The parameter FRAG_B4_RT representing fragmentation by hydrolysis has been replaced by the combination FRAG_SUBSTRATE RNA and FRAG_METHOD UR.

Cheers, Doc

the document provided is called README.txt, you can find it after decompressing the downloaded tarball file. It contains the command to get a list and descriptions for all available parameters of the Flux Simulator:

flux -t simulator —printParameters

or alternatively

flux -t simulator -o

Cheers, Doc

When I setting the parameter in my *.par file, some can not be recognized, such as FASTQ, FRAG_B4_RT. And it run correctly without these parameters.

[INFO] I am the Flux Toolbox (v1.0-RC5 build 569), nice to meet you!

[INFO] I am collecting information on the run.
[ERROR] Unable to load settings from homo.par

Error while parsing line 4. Parameter FASTQ not found. Check the spelling!

My system is:
Linux igenomics 2.6.32-32-server #62-Ubuntu SMP Wed Apr 20 22:07:43 UTC 2011 x86_64 GNU/Linux

And I'm runing FluxSimulator-1.0-RC5

Thanks very much

congratulations, your report led to the numerically interesting ticket 69 in the flux bugtracker! We will try to reproduce your error in RC5, to help us, could you please provide also the corresponding v1.0 parameter file that produced the error?

Best

I have a problem with read simulation. I have defined a .pro file where all transcripts have very similar abundance.

1:95302304-95320982C gene_123_iso_1 CDS 697 0.003577 114
1:95308664-95320982C gene_123_iso_2 CDS 670 0.003138 100
1:203830743-203839678W gene_1180_iso_1 CDS 942 0.003326 106
1:203832753-203839179W gene_1180_iso_2 CDS 355 0.003138 100
1:203830731-203839209W gene_1180_iso_3 CDS 967 0.003169 101
1:203830731-203839212W gene_1180_iso_4 CDS 532 0.003514 112
1:203830737-203839205W gene_1180_iso_5 CDS 478 0.002699 86
1:203830978-203834247W gene_1180_iso_6 CDS 248 0.002730 87

after library preparation I get read numbers that make me perfectly happy:

1:95302304-95320982C gene_123_iso_1 CDS 697 0.003577 114 0.00209560367060517 1747
1:95308664-95320982C gene_123_iso_2 CDS 670 0.003138 100 0.0017033527259641336 1420
1:203830743-203839678W gene_1180_iso_1 CDS 942 0.003326 106 0.002295927547531938 1914
1:203832753-203839179W gene_1180_iso_2 CDS 355 0.003138 100 0.001194745996521322 996
1:203830731-203839209W gene_1180_iso_3 CDS 967 0.003169 101 0.0022443471480837283 1871
1:203830731-203839212W gene_1180_iso_4 CDS 532 0.003514 112 0.001631380075571283 1360
1:203830737-203839205W gene_1180_iso_5 CDS 478 0.002699 86 0.0012043423499070354 1004
1:203830978-203834247W gene_1180_iso_6 CDS 248 0.002730 87 8.348827445570684E-4 696

but after sequencing I get this:

1:95302304-95320982C gene_123_iso_1 CDS 697 0.003577 114 0.00209560367060517 0.011101079589527092 1746
1:95308664-95320982C gene_123_iso_2 CDS 670 0.003138 100 0.0017033527259641336 0.0 0
1:203830743-203839678W gene_1180_iso_1 CDS 942 0.003326 106 0.002295927547531938 0.0 0
1:203832753-203839179W gene_1180_iso_2 CDS 355 0.003138 100 0.001194745996521322 0.0 0
1:203830731-203839209W gene_1180_iso_3 CDS 967 0.003169 101 0.0022443471480837283 0.0 0
1:203830731-203839212W gene_1180_iso_4 CDS 532 0.003514 112 0.001631380075571283 0.0 0
1:203830737-203839205W gene_1180_iso_5 CDS 478 0.002699 86 0.0012043423499070354 0.0 0
1:203830978-203834247W gene_1180_iso_6 CDS 248 0.002730 87 8.348827445570684E-4 0.0 0

most of the transcripts have no reads assigned.
What am I doing wrong?

This is my parameter file:

REF_FILE_NAME genes.gtf
PRO_FILE_NAME genes_expr_weak_bias1.pro
LIB_FILE_NAME genes_expr_weak_bias1.lib
SEQ_FILE_NAME genes_expr_weak_bias1.bed
GEN_DIR hg19/
NB_MOLECULES 20000000
EXPRESSION_K -0.6
EXPRESSION_X0 5.0E7
EXPRESSION_X1 9500.0
RT_MIN 10
RT_MAX 10000
FRAGMENTATION YES
LOAD_CODING YES
LOAD_NONCODING YES
FILTERING NO
READ_NUMBER 10000000
READ_LENGTH 75
PAIRED_END YES
TMP_DIR /tmp/global2/data_sim/
POLYA_SHAPE 2
POLYA_SCALE 300
ERR_FILE_NAME genes_expr_weak_bias1.err
RT_PRIMER RANDOM
FRAG_B4_RT YES
FRAG_MODE CHEMICAL
FRAG_LAMBDA 500.0
FASTQ NO
QTHOLD 0.0
FRAG_SIGMA 5.000000e-02
FRAG_THRESHOLD 1.000000e-01

I am using an older version of the flux simulator (built 20101223), because the new versions (4 and 5) died
during library generation with an null pointer exception:

[LIBRARY] Configuration
Rounds: 15
Mean: 0.5
Standard Deviation: 0.1

Processing Fragments * FAILED
[ERROR] Error while fragmenting : null
java.lang.NullPointerException
at fbi.genome.sequencing.rnaseq.simulation.fragmentation.Amplification.getGCcontent(Amplification.java:135)
at fbi.genome.sequencing.rnaseq.simulation.fragmentation.Amplification.process(Amplification.java:100)
at fbi.genome.sequencing.rnaseq.simulation.fragmentation.Fragmenter.process(Fragmenter.java:545)
at fbi.genome.sequencing.rnaseq.simulation.fragmentation.Fragmenter.call(Fragmenter.java:245)
at fbi.genome.sequencing.rnaseq.simulation.SimulationPipeline.call(SimulationPipeline.java:339)
at fbi.genome.sequencing.rnaseq.simulation.SimulationPipeline.call(SimulationPipeline.java:32)
at fbi.commons.flux.Flux.main(Flux.java:182)

any hints on that would also be helpful.

Thanks in advance,
Jonas

I filed your report in the issue tracker:

http://code.google.com/p/fluxcapacitor/issues/detail?id=67

Please find my answer there,

Best,

micha

I'm trying to find the latest simulator GUI and the sim executable file. However, all of the current downloads only contain the flux executable with the -t simulator option. However, I can't figure out how to launch a GUI.

I found a deprecated download (FluxSimulatr-v1.2) with the GUI but this is about 2 years old…I'm wondering how to launch the GUI on the newest version.

Thanks,

-jeremy

1:3044314-3044814W ENSMUST00000160944 NC 501 2.800345e-06 14 6.328407e-07 19 2.000235e-07 2
1:3092097-3092206W ENSMUST00000082908 NC 110 0.000000e+00 0 0.000000e+00 0 0.000000e+00 0
1:3456668-3503634W ENSMUST00000161581 NC 250 3.860476e-05 193 7.327630e-06 220 8.200963e-06 82

I expect the number of reads per transcript (which I got from the last column of the pro file) is proportional the transcript length (the 4th column), but it is not true..

I used the command: ./flux -t simulator -p mm9.par

here is the par file I used, some options I need to change?

1. average deviation from the annotated transcription start site (TSS)

#

1. number default: 25.0

TSS_MEAN 25.0

1. exponent of power-law underlying the expression profile

#

1. number default: -0.6

EXPRESSION_K -0.6

1. Target sequences file

#
SEQ_FILE_NAME mm9_simu.bed

1. number of RNA molecules initially in the experiment

#

1. number default: 5000000

NB_MOLECULES 5000000

1. Describes the method for subsampling fragments in order to meet the characteristics of the filter Distribution (see SIZE_DISTRIBUTION)
2. MH is a metropolis Hastings Filter
3. RJ is a rejection filter, picking the probability directly from the distribution
4. AC is a acceptance filter, picking the probability from the distribution,
5. but stretching it such that the probability of the 'most likely' element in the distribution is
6. stretched to 1.0.

#

1. [RJ, AC, MH] default: AC

SIZE_SAMPLING AC

1. The Genome directory

#
GEN_DIR mm9

1. Standard deviation value for GC distribution

#

1. 0.0<= number <= 100.0 default: 0.1

GC_SD 0.1

1. Maximum length observed after reverse transcription of full-length transcripts.

#

1. number default: 5500

RT_MAX 5500

1. GTF reference file

#
REF_FILE_NAME Mus_musculus.NCBIM37.63.updated.gtf

1. parameter controlling the exponential decay along the power-law

#

1. number default: 9500.0

EXPRESSION_X1 9500.0

1. Reverse transcription motif PWM.
2. This is disabled by default, but you can use a default matrix
3. by specifying 'default' as value.

#
RT_MOTIF default

1. Minimum length observed after reverse transcription of full-length transcripts.

#

1. number default: 500

RT_MIN 500

1. Switch on/off Reverse Transcription

#

1. true|false or yes|no default: true

RTRANSCRIPTION true

1. Error model file
2. You can use the default models '35' or '76' for the corresponding read lengths or
3. specify a custom error model file

#

1. [] default:

#

1. I chose not to supply an err file because the following error message I got:

#

1. [INFO] Reading error model /Users/wsun/bin/FluxSimulator-1.0-RC4/bin/sequence_err.txt
2. [ERROR] Unable to load error model : Not in GZIP format

#

1. ERR_FILE sequence_err.txt

1. Create .fasta/.fastq output. output.
2. If you specify an ERR_FILE as to be used as a quality model (or you use one of the default models)
3. the simulator will create a .fastq file with errors added to the actual sequence. If no error model is
4. specified, the simulator create a .fasta file where no error are added to the sequences.

#

1. true|false or yes|no default: false

FASTA true

1. The motif description for enzymatic digestion
2. You can specify a custom PWM file or
3. use one of the available defaults:
4. NlaIII or DpnII

#
FRAG_EZ_MOTIF NlaIII

#

1. [PDT, RH] default: RH

RT_PRIMER RH

1. Target library file

#
LIB_FILE_NAME mm9_simu.lib

1. Temporary directory

#
TMP_DIR /var/folders/Am/AmdRPmv5Fq8aIH-erB7Un++++TI/-Tmp-

1. PCR distribution file or 'default' to .
2. use a distribution with 15 rounds and 20 bins.
3. Set this to 'none' to disable amplification.

#

1. [] default: default

PCR_DISTRIBUTION default

1. determining the shape of the poly-A tail size distribution

#

1. 0.0<= number <= 1.7976931348623157E308 default: 2.0

POLYA_SHAPE 2.0

1. The Read length

#

1. number default: 36

READ_LENGTH 76

1. Always force RT

#

1. true|false or yes|no default: true

RT_LOSSLESS true

1. PCR duplication probability
2. This is used if GC filtering is disabled by setting GC_MEAN to NaN

#

1. 0.0<= number <= 1.0 default: 0.7

PCR_PROBABILITY 0.7

1. Method applied for Fragmentation
2. [NB] Nebulization fragmentation method.
3. [UR] Uniformal random fragmentation method.
4. [EZ] Enzymatic digestion as fragmentation method.

#

1. [NB, UR, EZ, NONE] default: UR

FRAG_METHOD UR

1. Parameter for the threshold on molecule length that cannot be broken by the shearfield during nebulization.

#

1. number default: 900.0

FRAG_NB_LAMBDA 900.0

1. geometry of the UR process (1=linear, 2=surface-diameter, 3=volume-diameter, etc.)

#

1. number default: NaN

FRAG_UR_DELTA NaN

1. Describes the distribution of fragments after filtering.
2. You can either specify an file with an empirical distribution, where each linerepresents the length of a read (no ordering required).
3. You can also specify a Normal-Distribution with mean and standard deviation using:
4. N(mean, sd)
5. for example: N(800, 200)

#

1. [] default:
2. SIZE_DISTRIBUTION 120hour_insertSizes.txt

#

1. true|false or yes|no default: true

LOAD_CODING true

1. Number of reads

#

1. number default: 5000000

READ_NUMBER 5000000

1. Mean value for GC distribution. Set this to 'NaN' to disable GC filtering.

#

1. 0.0<= number <= 1.0 default: 0.5

GC_MEAN 0.5

#

1. true|false or yes|no default: true

LOAD_NONCODING true

1. Parameter denoting the threshold on molecule population still breaking when determining convergence of iterative nebulizaiton.

#

1. number default: 0.1

FRAG_NB_THOLD 0.1

1. minimum length of fragments produced by UR fragmentation

#

1. 1.0<= number <= 1.7976931348623157E308 default: 1.0

FRAG_UR_D0 1.0

1. turn filtering on/off

#

1. true|false or yes|no default: false

FILTERING false

1. exhaustiveness of UR fragmentation, determining the number of breaks per unit length

#

1. number default: NaN

FRAG_UR_ETA NaN

1. parameter determining the maximum expression of the underlying power-law

#

1. number default: 5.0E7

EXPRESSION_X0 5.0E7

1. Target Profiler file

#
PRO_FILE_NAME mm9_simu.pro

1. controlling the average length of the poly-A tail sizes

#

1. 0.0<= number <= 1.7976931348623157E308 default: 300.0

POLYA_SCALE 300.0

1. Parameter specifying the substrate of fragmentation.

#

1. [DNA, RNA] default: DNA

FRAG_SUBSTRATE DNA

1. Pair end reads

#

1. true|false or yes|no default: false

PAIRED_END true

1. turn fragmentation on/off

#

1. true|false or yes|no default: true

FRAGMENTATION true

1. Parameter specifying the strength of the nebulization shearfield.

#

1. number default: 1.0

FRAG_NB_M 1.0

thanks a lot for your reply. I bypassed this problem by running fllux simulator on my laptop. I was having troubles on my desktop. So I am not sure whether this is something wrong with my system.

The GTF file I used is downloaded from

useast.ensembl.org/info/data/ftp/index.html

here are more of the error message:

[INFO] Checking GTF file
checking * Unsorted in line 220 - cannot perform gene clustering: 18 - ENSMUST00000122958 @ 3299448 after ENSMUST00000154135 @ 3337378
[GTF FILE] The GTF reference file given is not sorted, sorting it right now…
sorting GTF file java.util.concurrent.ExecutionException: java.io.IOException: No such file or directory
at java.util.concurrent.FutureTask$Sync.innerGet(FutureTask.java:222)
at java.util.concurrent.FutureTask.get(FutureTask.java:83)
at fbi.commons.tools.UnixStreamSorter.divide(UnixStreamSorter.java:295)
at fbi.commons.tools.UnixStreamSorter.sort(UnixStreamSorter.java:123)
at fbi.commons.tools.Sorter$1.call(Sorter.java:193)
at java.util.concurrent.FutureTask$Sync.innerRun(FutureTask.java:303)
at java.util.concurrent.FutureTask.run(FutureTask.java:138)
at java.util.concurrent.ThreadPoolExecutor$Worker.runTask(ThreadPoolExecutor.java:886)
at java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:908)
at java.lang.Thread.run(Thread.java:680)
Caused by: java.io.IOException: No such file or directory
at java.io.UnixFileSystem.createFileExclusively(Native Method)
at java.io.File.checkAndCreate(File.java:1704)
at java.io.File.createTempFile(File.java:1792)
at fbi.commons.file.FileHelper.createTempFile(FileHelper.java:993)
at fbi.commons.tools.UnixStreamSorter.sortAndWriteTempFile(UnixStreamSorter.java:363)
at fbi.commons.tools.UnixStreamSorter.access$200(UnixStreamSorter.java:33)
at fbi.commons.tools.UnixStreamSorter$2.call(UnixStreamSorter.java:275)
at fbi.commons.tools.UnixStreamSorter$2.call(UnixStreamSorter.java:272)
… 5 more
java.util.concurrent.ExecutionException: java.io.IOException: No such file or directory
at java.util.concurrent.FutureTask$Sync.innerGet(FutureTask.java:222)
at java.util.concurrent.FutureTask.get(FutureTask.java:83)
at fbi.commons.tools.UnixStreamSorter.divide(UnixStreamSorter.java:295)
at fbi.commons.tools.UnixStreamSorter.sort(UnixStreamSorter.java:123)
at fbi.commons.tools.Sorter$1.call(Sorter.java:193)
at java.util.concurrent.FutureTask$Sync.innerRun(FutureTask.java:303)
at java.util.concurrent.FutureTask.run(FutureTask.java:138)
at java.util.concurrent.ThreadPoolExecutor$Worker.runTask(ThreadPoolExecutor.java:886)
at java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:908)
at java.lang.Thread.run(Thread.java:680)
Caused by: java.io.IOException: No such file or directory
at java.io.UnixFileSystem.createFileExclusively(Native Method)
at java.io.File.checkAndCreate(File.java:1704)
at java.io.File.createTempFile(File.java:1792)
at fbi.commons.file.FileHelper.createTempFile(FileHelper.java:993)
at fbi.commons.tools.UnixStreamSorter.sortAndWriteTempFile(UnixStreamSorter.java:363)
at fbi.commons.tools.UnixStreamSorter.access$200(UnixStreamSorter.java:33)
at fbi.commons.tools.UnixStreamSorter$2.call(UnixStreamSorter.java:275)
at fbi.commons.tools.UnixStreamSorter$2.call(UnixStreamSorter.java:272)
… 5 more
java.util.concurrent.ExecutionException: java.io.IOException: No such file or directory
at java.util.concurrent.FutureTask$Sync.innerGet(FutureTask.java:222)
at java.util.concurrent.FutureTask.get(FutureTask.java:83)
at fbi.commons.tools.UnixStreamSorter.divide(UnixStreamSorter.java:295)
at fbi.commons.tools.UnixStreamSorter.sort(UnixStreamSorter.java:123)
at fbi.commons.tools.Sorter$1.call(Sorter.java:193)
at java.util.concurrent.FutureTask$Sync.innerRun(FutureTask.java:303)
at java.util.concurrent.FutureTask.run(FutureTask.java:138)
at java.util.concurrent.ThreadPoolExecutor$Worker.runTask(ThreadPoolExecutor.java:886)
at java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:908)
at java.lang.Thread.run(Thread.java:680)
Caused by: java.io.IOException: No such file or directory
at java.io.UnixFileSystem.createFileExclusively(Native Method)
at java.io.File.checkAndCreate(File.java:1704)
at java.io.File.createTempFile(File.java:1792)
at fbi.commons.file.FileHelper.createTempFile(FileHelper.java:993)
at fbi.commons.tools.UnixStreamSorter.sortAndWriteTempFile(UnixStreamSorter.java:363)
at fbi.commons.tools.UnixStreamSorter.access$200(UnixStreamSorter.java:33)
at fbi.commons.tools.UnixStreamSorter$2.call(UnixStreamSorter.java:275)
at fbi.commons.tools.UnixStreamSorter$2.call(UnixStreamSorter.java:272)
… 5 more

looks like a bug to me, but its hard to say. I already created a ticket in our bugtracker :

http://code.google.com/p/fluxcapacitor/issues/detail?id=64

Could you please post the full output produced by the simulator, especially the lines after

at java.util.concurrent.FutureTask$Sync.innerGet(FutureTask.java:222)

because this shows us where the error occurs in the simulator code. It would also help a lot if you could attach a version of the parameter file you are using to the ticket in the bugtracker. More importantly, could you attach a zipped version of the GTF file? A partial version of the GTF that we can use to reproduce the problem would also be fine.

If you have any further questions, please use the comment section at the ticket page. You can also add yourself to the cc list to stay informed about progress.

A possible workaround might be sorting the file manually, outside of a simulation. You can use the flux gtf sorter to try that:

flux -t sortGtf -i <yourGTF> -o <sortedGTF>

and then use the sorted file directly.

Best,
-thasso

[desktop:~/bin/FluxSimulator-1.0-RC4/bin] suninsky% ./flux -t simulator -p mm9.par
[INFO] I am the Flux Toolbox (v1.0-RC4 build 298), nice to meet you!

[INFO] No mode selected, executing the full pipeline (-x -l -s)
[INFO] I am collecting information on the run.

[INFO] Checking GTF file
checking * Unsorted in line 6 transcript id ENSMUST00000162897 used twice, on: 1,1
[GTF FILE] The GTF reference file given is not sorted, sorting it right now…
sorting GTF file java.util.concurrent.ExecutionException: java.io.IOException: No such file or directory
at java.util.concurrent.FutureTask$Sync.innerGet(FutureTask.java:222)

anyone knows what is wrong here?

Thanks a lot!

thank you for your detailed report, your observations look very much like a bug in the program—funny that it did not pop up before. I filed a corresponding ticket

New issue 62 by gmicha: error when producing FASTA / FASTQ
http://code.google.com/p/fluxcapacitor/issues/detail?id=62

we will proceed trying to reconstruct your scenario, by applying ensembl (v60) to hg19. To speed up the process, could you please provide us your parameter file? You could attach it to the bug report (URL above), or post it here.

Thanks,

micha

sequencing Problems reading 20: 336933, -3> 64286035 into 52: null
check for the right species/genome version!
java.lang.IndexOutOfBoundsException
at java.io.RandomAccessFile.readBytes(Native Method)
at java.io.RandomAccessFile.read(RandomAccessFile.java:338)
at java.io.RandomAccessFile.readFully(RandomAccessFile.java:397)
at fbi.genome.model.Graph.readSequence(Graph.java:1256)
at fbi.genome.model.bed.BEDobject2.readSequence(BEDobject2.java:251)
at fbi.genome.sequencing.rnaseq.simulation.Sequencer.createQSeq(Sequencer.java:565)
at fbi.genome.sequencing.rnaseq.simulation.Sequencer.access$1000(Sequencer.java:44)
at fbi.genome.sequencing.rnaseq.simulation.Sequencer$SequenceWriter.writeRead(Sequencer.java:888)
at fbi.genome.sequencing.rnaseq.simulation.Sequencer$Processor.process(Sequencer.java:737)
at fbi.genome.sequencing.rnaseq.simulation.Sequencer.sequence(Sequencer.java:290)
at fbi.genome.sequencing.rnaseq.simulation.Sequencer.call(Sequencer.java:107)
at fbi.genome.sequencing.rnaseq.simulation.SimulationPipeline.call(SimulationPipeline.java:351)
at fbi.genome.sequencing.rnaseq.simulation.SimulationPipeline.call(SimulationPipeline.java:32)
at fbi.commons.flux.Flux.main(Flux.java:168)

The names of the chromosome fasta files are equal to the chromosome names in the gtf file.
Do you have an idea what is going wrong here?

Best,
Thomas

 #MODEL 200 2500000 0 40 . 0.0 0.0 0.0015868 0.0015868 0.0015868 0.0015868 0.0015868 0.0015868 0.0015868 0.0015868 0.0022484 0.0022484 0.0022484 0.0022484 0.0022484 0.0022484 0.0040708 0.0040708 0.0040708 0.0040948 0.0040948 0.0040948 0.0043156 0.0072692 0.0072692 0.0110068 0.019494 0.0275796 0.0378608 0.0378608 0.0953624 0.4182544 0.4182544 0.4987388 1.0 1.0 1.0 1.0 1.0 1.0 1.0 

This yields the following stack trace:

 [INFO] I am the Flux Toolbox (v1.0-RC3 build 201), nice to meet you! [INFO] I am collecting information on the run. [INFO] Reading error model /home/joshua/experiments/104.flux_sim_new/err200.err.gz [ERROR] Unable to load error model : : only whitespace content allowed before start tag and not # (position: START_DOCUMENT seen \n#... @2:2) com.thoughtworks.xstream.io.StreamException: : only whitespace content allowed before start tag and not # (position: START_DOCUMENT seen \n#... @2:2) at com.thoughtworks.xstream.io.xml.XppReader.pullNextEvent(XppReader.java:67) at com.thoughtworks.xstream.io.xml.AbstractPullReader.readRealEvent(AbstractPullReader.java:126) at com.thoughtworks.xstream.io.xml.AbstractPullReader.readEvent(AbstractPullReader.java:119) at com.thoughtworks.xstream.io.xml.AbstractPullReader.move(AbstractPullReader.java:98) at com.thoughtworks.xstream.io.xml.AbstractPullReader.moveDown(AbstractPullReader.java:83) at com.thoughtworks.xstream.io.xml.XppReader.<init>(XppReader.java:37) at com.thoughtworks.xstream.io.xml.XppDriver.createReader(XppDriver.java:30) at com.thoughtworks.xstream.io.xml.XppDriver.createReader(XppDriver.java:34) at com.thoughtworks.xstream.XStream.fromXML(XStream.java:789) at fbi.genome.sequencing.rnaseq.simulation.error.MarkovErrorModel.loadErrorModel(MarkovErrorModel.java:325) at fbi.genome.sequencing.rnaseq.simulation.Sequencer.loadErrors(Sequencer.java:208) at fbi.genome.sequencing.rnaseq.simulation.SimulationPipeline.call(SimulationPipeline.java:306) at fbi.genome.sequencing.rnaseq.simulation.SimulationPipeline.call(SimulationPipeline.java:32) at fbi.commons.flux.Flux.main(Flux.java:168) Caused by: org.xmlpull.v1.XmlPullParserException: only whitespace content allowed before start tag and not # (position: START_DOCUMENT seen \n#... @2:2) at org.xmlpull.mxp1.MXParser.parseProlog(MXParser.java:1519) at org.xmlpull.mxp1.MXParser.nextImpl(MXParser.java:1395) at org.xmlpull.mxp1.MXParser.next(MXParser.java:1093) at com.thoughtworks.xstream.io.xml.XppReader.pullNextEvent(XppReader.java:52) ... 13 more [ERROR] Unable to load error model : : only whitespace content allowed before start tag and not # (position: START_DOCUMENT seen \n#... @2:2) 

Has the spec for error files changed? The stack trace makes it look like you guys are using a pulldom parser that expects an XML file. Any other clues would be much appreciated.

Thanks & best,
Josh

if I understand correctly, you want to filter fragments according to their insert size from the library before sequencing. This is realized by a size selection step, which you can turn on by setting parameter $\tt{FILTERING}$ to $\tt{YES}$. You can control the size filtering by providing as parameter $\tt{SIZE_DISTRIBUTION}$ either a file describing an empirical distribution, e.g., the file $\tt{expAll.isizes}$ in the program bundle, or a parameterized normal distribution, e.g., as described in the file $\tt{README.txt}$ a string $\tt{N(200,20)}$ describes a gaussian distribution with mean 200nt and standard-deviation 20nt. A hard on/off transition at the boundaries 50 and 500 is probably not very realistic as usually filtering is performed by gel segregation, however, it would be possible to construct empiric distributions where bins <50 and >500 have a chance of 0 to pass filtering. Finally, also the subsampling method, parameter $\tt{SIZE_SAMPLING}$, can bias the representation of the insert size distribution.

Best,

micha

we want to provide more information on the UR algorithm soon. You can control the average fragment size with Eta, set it to a value close to the average size you aim for. Delta controls the spread of the fragment size distribution, when deactivated (NaN) it is set dynamically for each transcript according to physical attributes.

Best,

micha

I'm interested in simulating a paired-end dataset, with uniform random fragmentation of transcribed cDNAs to an average size of about 200 nucleotides, and am wondering how to set the fragmentation parameters Delta and Eta to achieve this.

When you get a moment, please let me know!
Thanks,
Josh

thanks,

maria

actually the strand information is currently in the read IDs you get out of the simulator, which look like

chrX:1-9786W:F00123456:3:9786:5319:5513:5319:S/1

where chrX is the chromosome ID, 1-9786 is the genomic location, DNA strand of the transcript (C=Crick, W=the other one), F00123456 is the transcript ID of the RNA molecule, then follows some info on the fragment has been sequenced (\:[0-9]+), and finally-what I think you are interested in-the read orientation with respect to the directionality of transcription of the RNA molecule (S=sense, A=anti-sense), eventually followed by a mate pair identifier (/1 or /2) if paired-end sequencing is turned on.

Unlike reality, the strand assignment is loss-less, i.e., the simulation does not loose the strand in some molecules. However, that could be easily mimicked by a post-processing step that disregards strand information for a corresponding fraction of reads.

All the best,

micha

That said, the RNA-seq protocols I use produce strand-specific libraries, where the sequence as read corresponds to the plus strand of the RNA. This is particularly useful for assembling known transcripts, as well as identifying the strands of novel transcribed regions. Is there an option I can set in Flux Simulator to get it to produce a strand-specific library? This would be incredibly useful.

Thanks & please let me know!

Best,
Josh

Bests,
Marcel

it is possible to completely disable the TSS_MEAN feature by setting the value to "NaN". Unfortunatly there is a bug in the settings validation ( http://code.google.com/p/fluxcapacitor/issues/detail?id=55 ). It will be fixed soon and I will update the package on the website.

cheers,

thasso

Thanks for a reply and for the cool tool,
Marcel

i have another question. do you know anyway to go from the .bed results to a .sam/.bam file in R?, thanks!

maria

the problem was of the .gtf, i tried to use just a part of the genome (just to try it and i didnt do it right). but i have another question. Now is about how to create the "fastq" file. Im using only one human chromosome (to start with) and ive download the .fa file of that chromosome (only the one for that chromosome, maybe it needs all) and also set the "FASTQ" variable to TRUE in the .par file. The thing is that if I set flux simulator to only generate the .bed file it doesnt give any error but if i ask for the .fastq file it doesnt do anything it says "initing…" and it stays there forever… do you know which could be the problem??

thank you very much!

maria

does anyone have an error file for a read length around 75 bp, and is willing to share this with me??
I tried generating one on my own, but I had some troubles with that.

Thank you very much,
Andi

seems like the symptoms reported in Issue52, please try to report problems of technical nature there. Note that we uploaded a largely redesigned simulator version (1.0 RC1), which also excludes parallelization that seemed to cause JVM/OS-dependent crashes or deadlocks. I will try to update the webpages with new documentation ASAP, for the moment please refer to the readme file bundled in the tarball. Also, additional logging depth is provided by the —log DEBUG parameter, please post a corresponding stderr output with your bug report if the problem persists for you.

cheers

RNA fragments can be lost due to non-successful RT, cDNA molecules can be lost during an (optional) size filtering step. Therefore the number of molecules in the final library is not always a fixed factor >1 of the initial molecule count.

Best

actually it is quite straightforward to include homozygote mutations in the simulator, by just changing the corresponding nucleotides in the genomic sequence file. Heterozygote mutations are a bit more tricky, as they require

(a) a format to provide alternative nucleotides at the same chromosomal position

(b) a model for allelic frequencies during gene expression

Concerning (a), we thought about optionally providing a VCF (Variant Call Format) file as they are adopted for the 1000 genomes project along with the genomic reference. Point (b) is far more complex, but maybe initially a 50/50 assumption is fine for some studies.

I filed correspondingly request for enhancement 52

Best, micha

seems like the symptoms reported in Issue52, please try to report problems of technical nature there. Note that we uploaded a largely redesigned simulator version (1.0 RC1), which also excludes parallelization that seemed to cause JVM/OS-dependent crashes or deadlocks. I will try to update the webpages with new documentation ASAP, for the moment please refer to the readme file bundled in the tarball. Also, additional logging depth is provided by the —log DEBUG parameter, please post a corresponding stderr output with your bug report if the problem persists for you.

cheers

program crashes should better be discussed in the issue tracker, the described one may be related to the Issue48 or a new report may be opened. Please note that we uploaded a largely redesigned simulator version (version 1.0 RC1) with additional logging depth is provided by the —log DEBUG parameter, please post with your bugreport a corresponding stderr output if the problem persists for you.

let $reads_i$ be the number of reads you sampled during sequencing from transcript $i$ (see read ID), and $length_i$ it's length in number of nucleotides, then you can calculate the corresponding $RPKM_i$ value by

where $\textrm{READ_NR}$ is the number of reads in the experiment, i.e., the number of reads you find in the .BED file in case you are not simulating your own sample contaminations in the .GTF file you provide.

The FPKM value can be obtained very similarly, by counting every fragmenteither identified by mate pairs or by the corresponding fragment number in the read identifierjust once.

Best

with the parameter POLYA_SCALE you can influence the length of the simulated poly-A tails, with the parameter POLYA_SHAPE their distribution. If the transcripts in your test file are particularly short, try changing POLYA_SCALE to lower values, depending on your sample size (i.e., the number of transcripts in your test file) may choose to get the distribution tighter by raising the POLYA_SHAPE parameter.

Best

the file you provide for the parameter PRO_file_name is written first after reading the input annotation (the .GTF file), the library (.LIB) file is written after library construction (RT and fragmentation), and the .BED file after sequencing. I did not understand what you mean by you created these files yourself—did you create empty files or the directories where the corresponding output is to be written?

Best

micha

Im trying to generate simulated sequencing data with your FluxSimulator. As I understand your manual, to do the annotation we only should need the .GTF file, which ive created (it’s from your drososet.gtf, just a part of it), and the “annotation” step would create the “PRO_file_name”, but it doesn’t and it actually stop working. I have changed the .par file and set my files in the path variables. In fact, if I create the pro file myself (and also all the rest bed, and lib files) it doesn’t give any problems, but as I understand those files are the ones that FluxSimulator should create, isn’t it?

Thank you very much,

maria

When I run the Flux Simulator GUI to sequence some reads, after some time, I get the following error in the command line:
java.lang.ArrayIndexOutOfBoundsException
at java.lang.System.arraycopy(Native Method)
at genome.sequencing.rnaseq.simulation.A.A(Unknown Source)
at genome.sequencing.rnaseq.simulation.A.A(Unknown Source)
at genome.sequencing.rnaseq.simulation.A$_A.A(Unknown Source)
at genome.sequencing.rnaseq.simulation.A$_A.run(Unknown Source)
at genome.sequencing.rnaseq.simulation.A.x(Unknown Source)
at genome.sequencing.rnaseq.simulation.A.run(Unknown Source)
at genome.sequencing.rnaseq.simulation.A.A.run(Unknown Source)
at java.lang.Thread.run(Unknown Source)
at genome.sequencing.rnaseq.simulation.A.E$_A.run(Unknown Source)

The previous steps work properly and I get the outputs and plots. It's just this sequencing step.
I also have "Enable Fasta/Fastq file" selected.

It is the same situation in the command line; all the step but the sequencing step work, except that there, it does not even generate an error, and just terminates!

READ_LENGTH 50
READ_NUMBER 2000000
initing

I just have one question: is it possible to simulate also SNP and DIP mutations? For the project I am working on this would be very interessting.

Regards Andi

[SEQUENCING] getting the
reads
PRO_FILE_NAME /data1/bsi/algorithm_dev/m087494_algorithm_dev/Development/FluxSimulator/demo/drosotest.pro
SEQ_FILE_NAME /data1/bsi/algorithm_dev/m087494_algorithm_dev/Development/FluxSimulator/demo/drosotest.bed
READ_LENGTH 100
READ_NUMBER 200000000
initing
501682308 lines submitted
zipping ***
501682308 lines zipped
java.util.zip.ZipException: error in opening zip file
at java.util.zip.ZipFile.open(Native Method)
at java.util.zip.ZipFile.<init>(ZipFile.java:114)
at java.util.zip.ZipFile.<init>(ZipFile.java:131)
at genome.sequencing.rnaseq.simulation.A.g(Unknown Source)
at genome.sequencing.rnaseq.simulation.A.run(Unknown Source)
at genome.sequencing.rnaseq.simulation.FluxSimulator.G(Unknown Source)
at genome.sequencing.rnaseq.simulation.FluxSimulator.run(Unknown Source)
at genome.sequencing.rnaseq.simulation.FluxSimulator.main(Unknown Source)

PRO_FILE_NAME /Users/zh/Downloads/FluxSimulator/chr1/test_chr1.pro
LIB_FILE_NAME /Users/zh/Downloads/FluxSimulator/chr1/test_chr1.lib
SEQ_FILE_NAME /Users/zh/Downloads/FluxSimulator/chr1/test_chr1.bed
GEN_DIR /Users/zhaohao/Downloads/FluxSimulator/chr1
TMP_DIR /var/folders/fY/fY+RKWXZHrqzw9LIcH2+3U+++TU/-Tmp-
NB_MOLECULES 5000000
EXPRESSION_K -0.6
EXPRESSION_X0 5.0E7
EXPRESSION_X1 9500.0
TSS_MEAN 25.0
POLYA_SHAPE 2.0
POLYA_SCALE 300.0
RT_MIN 500
RT_MAX 5500
RT_PRIMER RANDOM
FRAGMENTATION NO
FRAG_B4_RT NO
FRAG_MODE PHYSICAL
FRAG_LAMBDA 900.0
FRAG_THRESHOLD 0.1
FILTERING NO
LOAD_CODING YES
LOAD_NONCODING YES
FILT_MIN 200
FILT_MAX 250
READ_NUMBER 5000000
READ_LENGTH 75
PAIRED_END YES
FASTQ NO