generating simulated RNA-Seq data by using flux simulation

Question

[stackunderflow]

I have tried to use flux simulation tool to generate simulated RNA-seq data.

I gave the following parameter file to flux-simulation shell script

## File locations

REF_FILE_NAME   cElegansAnnotation.gtf
GEN_DIR         chromFa

## Library preparation
# Expression
NB_MOLECULES    5000000
TSS_MEAN    50
POLYA_SCALE     100
POLYA_SHAPE     1.5

# Fragmentation
FRAG_SUBSTRATE  RNA
FRAG_METHOD UR
FRAG_UR_ETA     350

# Reverse Transcription
RTRANSCRIPTION  YES
RT_MOTIF    default

# Amplification 
PCR_DISTRIBUTION default
GC_MEAN      NaN
PCR_PROBABILITY  0.05

# Size Filtering
FILTERING   NO


## Sequencing
READ_NUMBER 1000000
READ_LENGTH 100
PAIRED_END  YES

# create a fastq file
FASTA           YES

According to this parameter, flux-simulation should have given reads with length 100. However when I look at the output, I have seen that reads with less then length of 100 are also included in my fasta file:

>chrI:47472-49416W:Y48G1C.12:3:651:351:594:A/1
CGTCGAAATTAGTGATATTTTTATCGGGAATCGGTCCGTGTGGTTCTCCGGTGAATATTCGATTCGTTGTGGAGACACGAGATCGCTGGGGTCCAAGGAC
>chrI:47472-49416W:Y48G1C.12:4:651:394:467:S/1
TACGCGACAAAAATGGGAAACCGAATCGCGTTTTTTGGCTTCAAGTACAAGTTATTCAGAATCATCAAAATGGG
>chrI:47472-49416W:Y48G1C.12:4:651:394:467:A/2
CCCATTTTGATGATTCTGAATAACTTGTACTTGAAGCCAAAAAACGCGATTCGGTTTCCCATTTTTGTCGCGTA
>chrI:47472-49416W:Y48G1C.12:5:651:126:142:S/2
AGTTGTAAAAGCGGATT

So, is there anyone who can help me to fix it?

[Devon Ryan]

Have you compared your settings to one of the examples? Presumably the RT_MIN setting would prevent what you're seeing.

[stackunderflow]

Thans for reply, Yes I prepared my parameter file based on the link you provided. I do not give any RT_MIN to my file because its default value looks like 500 in the link?Isn't it

[Devon Ryan]

It's unclear whether that's the default or the value they specified. In either case, give it a try.

[k.kathirvel93]

Hi stack,

I hope you have finished this thread. But now i have started. Yes am using flux to simulate illumina RNA-seq paired-end data. Can you tell me how can i separate left and Right ends? and which one is sense and Antisense A/1 or S/2?. and i am also getting some reads which are below 35nt. will you solve.