polyA imapct on downstrem alaysis particularly alternative splicing
After trimming my RNA-seq reads, FastQC still shows polyA sequences at the 3′ end.
Could this remaining polyA affect downstream analysis, particularly alternative splicing detection (e.g., splice junction identification)?
Also, is it necessary to explicitly provide a polyA sequence file or use a dedicated polyA trimming step to remove these sequences before alignment, or can splice-aware aligners like STAR handle this adequately?
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That indicates that you have not done the trimming properly. While
trimmomaticshould be able to handle this, in case you are not able to find the right option combination, you could use alternate programs likefastporbbduk(from BBMap suite) to trim poly-A.Aligners should soft-clip any part of the read that does not align. So in theory, STAR should be able to handle these poly-A sequences.
I am attaching my FastQC report after trimming. There is still some polyA enrichment visible at the 3′ end of the reads. Based on the level shown in the report, would you consider this amount of residual polyA substantial enough to potentially affect downstream alternative splicing analysis, or is this generally within an acceptable range?
While the scale is unclear the residual polyA should not affect anything. Use one of the other programs mentioned above, if you want to remove the residual polyA that remains.
show us a sequence that was not trimmed properly
java -jar /home/moradi/app/Trimmomatic-0.39/trimmomatic-0.39.jar PE -threads 8 \ SRR31111525_1.fastq SRR31111525_2.fastq \ SRR31111525_R1.trimPE.fq.gz SRR31111525_R1.trimUn.fq.gz \ SRR31111525_R2.trimPE.fq.gz SRR31111525_R2.trimUn.fq.gz \ ILLUMINACLIP:/home/moradi/app/Trimmomatic-0.39/adapters/TruSeq3-PE.fa:2:30:10 \ SLIDINGWINDOW:7:25 \ MINLEN:50