Quality Control of 454 RNA seq data

[Dr. Deepak]

Dear Friends
I am new to NGS technology. I have 454 single end RNA seq data. for This i had used trimmomatic, fastx and qtrim tool for quality control. Below is my pipeline for quality filtering and final fastqc output. Although, the per base sequence quality graph shows the best quality but the fastqc option is still mark X sign. I am also not happy with the per base GC content and sequence content. Could anybody suggest that is the final data is ok for assembly or i need more quality filter.

Any help will be appreciated.
Thanks

My Pipeline Commands are

java -jar /opt/software/Trimmomatic-0.32/trimmomatic-0.32.jar SE SRR1646514.fastq SRR1646514_filter1.fastq ILLUMINACLIP:/opt/software/Trimmomatic-0.32/adapters/TruSeq2-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36 HEADCROP:15
~/software/fastx_toolkit_0.0.13/fastx_trimmer -Q 33 -m 50 -t 9 -i SRR1646514_trimmomatics_filter.fastq -o SRR1646514_trimmomatics_filter_t_9_m_50.fastq
QTrim_v1_1/QTrim_v1_1 -m 30 -mode 2 -l 50 -out_format 2 -seq_id_stat -plot pdf -fastq ~/Desktop/SRR1646514_trimmomatics_filter.fastq

The final ourput of the high qulaity reads in fastQC graph is here.
http://www.dropbox.com/s/h6q97psk3ga...stqc.html?dl=0

[Brian Bushnell]

Cross-posted:

https://www.biostars.org/p/152932/