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i still got an error while preparing transcript sequence d= uring 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 an= d 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: 1.30-20140614=
022955)
[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 perfor=
m 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 sort=
ed version.
[GTF FILE] The Simulator will use /mountpoints/wntshare/project/aczerny/Un=
titled_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/p=
roject/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, ou=
t 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 72=
1805
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: p=
os -1, len 124,
check whether chromosomal sequence exists / has the correct size
java.lang.RuntimeException: Problems reading sequence chr1: pos -1, len 12=
4,
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.getMap=
TxSeq(Fragmenter.java:381)
at barna.flux.simulator.fragmentation.Fragmenter.proces=
s(Fragmenter.java:545)
at barna.flux.simulator.fragmentation.Fragmenter.call(F=
ragmenter.java:264)
at barna.flux.simulator.SimulationPipeline.call(Simulat=
ionPipeline.java:440)
at barna.flux.simulator.SimulationPipeline.call(Simulat=
ionPipeline.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 12=
4,
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.getMap=
TxSeq(Fragmenter.java:381)
at barna.flux.simulator.fragmentation.Fragmenter.proces=
s(Fragmenter.java:545)
at barna.flux.simulator.fragmentation.Fragmenter.call(F=
ragmenter.java:264)
at barna.flux.simulator.SimulationPipeline.call(Simulat=
ionPipeline.java:440)
at barna.flux.simulator.SimulationPipeline.call(Simulat=
ionPipeline.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/proje=
ct/aczerny/Untitled_Folder/parameters.lib not found. Make sure fragmentatio=
n was done !
at barna.flux.simulator.SimulationPipeline.call(Simulat=
ionPipeline.java:450)
at barna.flux.simulator.SimulationPipeline.call(Simulat=
ionPipeline.java:54)
at barna.commons.launcher.Flux.main(Flux.java:197)