Hello,
I have the following question on paired end reads from RNA-seq:
To get the total number of obtained reads from paired end reads (2 fastq files are generated (R1, R2), is it correct to add the read count derived from R1 and R2?
Thank you very much for your clarification
Yes. For example for the genome coverage calculation you count each read as if coming from separate fragment. Or even simpler you worry yourself about the number of bases sequenced.
The best way is to trim and filter the files and after to map on your genome to produce a bam file that will give you the real counts, avoiding counting bias and false results. but at the first point is how to count what you don't know? reading needs positions and therefore aligned data..
fred
Tamara Salloum
First sequencing can either be single-end where each sample has only sequence data or paired-end where each sample has two sequence data (R1 and R2).
The steps below can be used if your data is your data is RNA-seq.
The first step is to perform QC using tools like FastQC to help you understand your data
Then you trim your reads to remove adapters,bad reads,etc. Then you map the trimmed reads (R1 and R2) them onto a reference genome using tools like HISAT2. A single bam file will be generated.
Then you obain the read counts from the bam file. (Tools such as feature count, HTSeq, stringtie).
Then the read counts can be normalized depending on your research objectives.
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