I'm new to RNA-seq data analysis..
I downloaded 6 samples (3 for every condition) and I ran QC on them and had to trim the adaptors and poor reads.. what happened is that 3 replicates of one condition received (aggressive trimming) and removed around 15% of reads as an average. while 7% of reads have been thrown from the other condition replicates.
now removal of 15% will affect my downstream analysis, so is there any recommendations to improve the trimming procedure or this normal because of issues in the original row data ??
my trimmomatic command:
java -jar ~/Desktop/Trimmomatic-0.36/trimmomatic-0.36.jar PE -phred33 SRR11771_1.fastq SRR11771_2.fastq TRMD_SRR11771_1_paired.fastq TRMD_SRR11771_1_unpaired.fastq TRMD_SRR11771_2_paired.fastq TRMD_SRR11771_2_unpaired.fastq ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
my plan is to do exon-centric analysis using tophat and star as aligners and miso, mats and suppa for differential splicing analyses..
Your help is really appreciated.
15% is not massive (unless you have few reads to begin with) :-)
Your command conflates adapters and quality, so it's impossible to determine why the reads are being trimmed. I suggest you first do adapter-trimming, and then possibly do quality trimming. In general, quality-trimming decreases the accuracy of alignment, though trimming to a low level like Q6 can sometimes be beneficial.
Thank you all for sharing the useful information with me..
I will take into account the different ideas posted here.
in the RNAseq data analysis, You have to be careful to strike a balance between acceptable quality and also minimize the number of discarded reads. it should be noted, all the adapters contamination should be trim. I recommend you 123Fastq which combine FASTQC and trimmomatic in a highly interactive graphical user interface. it also added some improvements to QC modules of FASTQC, added a Kmer-based approach to remove adapters in the trimming, and many other features. try it your own: https://sourceforge.net/projects/project-123ngs/
Instead of posting this in multiple old threads it would be best to post an independent
toolspost one time. That would be proper way of announcing your software.