Stil an error while preparing sequences

 i still got an error while preparing transcript sequence during PCR distribution:

I just want to get a simple human experiment with 2 groups 3 replicates (fastq files) each. Some of them should be even expressed between groups and some should be highly different expressed. How can i acomplish this?

My genome: Homo_sapiens.GRCh37.73.dna.primary_assembly.fa

My gtf:gencode.v18

the key error, i think:

and i dont want to alter my genome file, because the position wont match anymore with the gtf-file:

[INFO] Loading default PCR distribution

    preparing transcript sequences  ERROR

[ERROR] Error while preparing sequences: Problems reading sequence chr1: pos -1, len 124,

check whether chromosomal sequence exists / has the correct size

[INFO] Flux-Simulator v1.2.2-20140614022955 (Flux Library: 1.30-20140614022955)

[INFO] No mode selected, executing the full pipeline (-x -l -s)
[INFO] I am collecting information on the run.
    initializing profiler  **********
[CAUTION] I overwrite the expression values in file parameters.pro, please confirm:
    (Yes,No,Don't know) Yes

[INFO] Reading error model 76 bases model
[WARN] The error model supports a read length of 76 but
you are trying to create reads of length 36. We are scaling.

[INFO] Checking GTF file
    Checking GTF *[WARN] Unsorted in line 5 - cannot perform gene clustering: chr1 + ENST00000591440.1 @ 719750 after ENST00000590848.1 @ 728262
    Checking GTF ********** OK (00:00:00)
[GTF FILE] The GTF reference file given is not sorted, but we found a sorted version.
[GTF FILE] The Simulator will use /mountpoints/wntshare/project/aczerny/Untitled_Folder/filtered_gtf_v18_sorted.gtf
[GTF FILE] You might want to update your parameters file
[PROFILING] I am assigning the expression profile
    Checking GTF ********** OK (00:00:00)
    Reading reference annotation  OK (00:00:00)
    found 144 transcripts

[PROFILING] Parameters
    NB_MOLECULES    100000
    EXPRESSION_K    -0.6
    EXPRESSION_X0    9500.0
    EXPRESSION_X1    9.025E7
    PRO_FILE_NAME    /mountpoints/wntshare/project/aczerny/Untitled_Folder/parameters.pro

    profiling ********** OK (00:00:00)
    Updating .pro file  ********** OK (00:00:00)
    molecules    99996

[LIBRARY] creating the cDNA libary
    Initializing Fragmentation File ********** OK (00:00:00)
    99996 mol initialized
[LIBRARY] Fragmentation UR
[LIBRARY] Configuration
        D0: 1.0
        Delta:  Not specified, depends on sequence length
        Eta: 223.09448802182916

    Processing Fragments ********** OK (00:00:01)
        721945 mol: in 99996, new 621949, out 721945
        avg Len 201.0221, maxLen 572
[LIBRARY] Reverse Transcription
[LIBRARY] Configuration
        Mode: RH
        PWM: No
        RT MIN: 500
        RT MAX: 5500

    Processing Fragments ********** OK (00:00:02)
        721945 mol: in 721945, new 0, out 721805
        avg Len 175.60474, maxLen 562
        start amplification
[INFO] Loading default PCR distribution
    preparing transcript sequences  ERROR
[ERROR] Error while preparing sequences: Problems reading sequence chr1: pos -1, len 124,
check whether chromosomal sequence exists / has the correct size
java.lang.RuntimeException: Problems reading sequence chr1: pos -1, len 124,
check whether chromosomal sequence exists / has the correct size
    at barna.model.Graph.readSequence(Graph.java:721)
    at barna.model.Graph.readSequence(Graph.java:608)
    at barna.model.Graph.readSequence(Graph.java:430)
    at barna.model.Transcript.getSplicedSequence(Transcript.java:451)
    at barna.flux.simulator.fragmentation.Fragmenter.getMapTxSeq(Fragmenter.java:381)
    at barna.flux.simulator.fragmentation.Fragmenter.process(Fragmenter.java:545)
    at barna.flux.simulator.fragmentation.Fragmenter.call(Fragmenter.java:264)
    at barna.flux.simulator.SimulationPipeline.call(SimulationPipeline.java:440)
    at barna.flux.simulator.SimulationPipeline.call(SimulationPipeline.java:54)
    at barna.commons.launcher.Flux.main(Flux.java:197)
Caused by: java.lang.NullPointerException
    at barna.model.Graph.getRAF(Graph.java:1198)
    at barna.model.Graph.readSequence(Graph.java:651)
    ... 9 more
 FAILED
[ERROR] Error while fragmenting : Problems reading sequence chr1: pos -1, len 124,
check whether chromosomal sequence exists / has the correct size
java.lang.RuntimeException: Problems reading sequence chr1: pos -1, len 124,
check whether chromosomal sequence exists / has the correct size
    at barna.model.Graph.readSequence(Graph.java:721)
    at barna.model.Graph.readSequence(Graph.java:608)
    at barna.model.Graph.readSequence(Graph.java:430)
    at barna.model.Transcript.getSplicedSequence(Transcript.java:451)
    at barna.flux.simulator.fragmentation.Fragmenter.getMapTxSeq(Fragmenter.java:381)
    at barna.flux.simulator.fragmentation.Fragmenter.process(Fragmenter.java:545)
    at barna.flux.simulator.fragmentation.Fragmenter.call(Fragmenter.java:264)
    at barna.flux.simulator.SimulationPipeline.call(SimulationPipeline.java:440)
    at barna.flux.simulator.SimulationPipeline.call(SimulationPipeline.java:54)
    at barna.commons.launcher.Flux.main(Flux.java:197)
Caused by: java.lang.NullPointerException
    at barna.model.Graph.getRAF(Graph.java:1198)
    at barna.model.Graph.readSequence(Graph.java:651)
    ... 9 more



[ERROR] Fragmentation file /mountpoints/wntshare/project/aczerny/Untitled_Folder/parameters.lib not found. Make sure fragmentation was done !
java.lang.RuntimeException: Fragmentation file /mountpoints/wntshare/project/aczerny/Untitled_Folder/parameters.lib not found. Make sure fragmentation was done !
    at barna.flux.simulator.SimulationPipeline.call(SimulationPipeline.java:450)
    at barna.flux.simulator.SimulationPipeline.call(SimulationPipeline.java:54)
    at barna.commons.launcher.Flux.main(Flux.java:197)