Read groups in RNA-seq

Question

[Petesview]

Hi bioinformaticians,

If I have paired end reads for my samples and I am looking to perform alignment either using HISAT2 or STAR, will the alignment results change significantly depending on whether the reads are separately aligned based on read groups (merged subsequently), or if I do it in a single alignment for all read groups of the same sample?

I understand that some tools such as Picard and GATK require read group information in the input. But outside of this application, is there a big difference between the two processing procedures?

Furthermore, if I have read_1.fq and read_2.fq files for a given sample, does this intuitively indicate that my paired end reads from the sample are of the same read group?

[biofalconch]

Just to understand, by read groups you mean technical replicates? (same biological sample, different sequencing run)

[Petesview]

According to the definition, read group is “a set of reads that are generated from a single run of sequencing instrument”. So I believe for a single replicate, reads that are generated from different runs of sequencing?

[biofalconch]

Ok, so pooling samples is fine as far as I know as long as they are technical replicates from the same biological replicate. Also I coulnd't really understand the second question, what do you mean by indicate they are from the same read group?

[Petesview]

I was just wondering if I have read_1.fq and read_2.fq, should I just consider these as being sequenced from the same experiment run?

[GenoMax]

Are _1 and _2 files of reads from a read pair? Generally R1/R1 (or _1/_2) will be used to reflect that. They will be sequenced in the same run.