This is a test version of Biostars. For the public version, visit https://www.biostars.org.
Quality of RNA Seq data

Hello,

I am analyzing human RNA Seq data from a paper (PRJNA421274). I did FastQC on the RAW data and it showed high difference between %A and %T as shown in the link below

https://ibb.co/vX6kL95

Even after trimming it is the same.Is it unsusal and should i use this samples? Is there a way to reduce this difference? I am new to RNA Seq Analysis.Any help is appreciated!

Thank you in advance.

rna-seq fastqc sequencing rna-seq
• 1,007 views • link
written by ashwin.koppayi • ashwin.koppayi

Don't bother too much with fastqc. Trim adapters and align the data, then see if you get a good mapping rate and if most reads align to exons, that is the more relevant QC than fastqc itself.

• 0 views • link
written by ATpoint ATpoint

Thank you for the prompt response. I aligned trimmed samples using STAR using the default options but number of uniquely mapped reads is quite low less than 10% and high %unmapped:too short. I tried to tweak this as suggested in (https://github.com/alexdobin/STAR/issues/169) but it is still too low.

• 0 views • link
written by ashwin.koppayi • ashwin.koppayi

How did you trim?

• 0 views • link
written by ATpoint ATpoint

I used Trimmomatic

java -jar /export/share/apps/Trimmomatic-0.32/trimmomatic-0.32.jar PE -threads 16 SRR6350434_1.fastq.gz SRR6350434_2.fastq.gz  ~/files/trim/SRR6350434_1_paired.fastq.gz  ~/files/trim/SRR6350434_1_unpaired.fastq.gz ~/files/trim/SRR6350434_2_paired.fastq.gz ~/files/trim/SRR6350434_2_unpaired.fastq.gz ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
• 0 views • link
updated by Ram Ram • written by ashwin.koppayi • ashwin.koppayi

0 answers

No answers yet.

Log in to answer this question.

More posts like this