Paired-end reads

Question

[kiran]

Hi, i'm new to Aligning tools and handling DNA data.

I understood the process of aligning the reads to reference genome and qualifying them based of quality. (i was interpreting results from BAM file). but i'm very confused about the paired end reads. i'm assuming that we have reads in FASTQ file in this way. say 150bp sequence and it is splitted into 2 reads, Read 1 and Read 2. and also we have reverse compliment of same Read 1 and Read 2. is it true ?

i have gone through some materials but still confused about this, could you please give me clarification about this.

Thanks for your time.

Answer 1

[GenoMax]

Key thing to keep in mind is you are sampling/sequencing a fragment of DNA (from a library of such fragments made from a sample) from both ends. Since sequencing is always done in 5'-->3' direction, reads in R1/R2 files come from two strands of fragments. Sequenced fragments are generally longer than the read lengths so the two reads do not overlap in the middle. In some special cases insert sizes may be deliberately made smaller to have the reads overlap.

Images included in these past threads should make this clear:
Some explanation about what a paired-end sequencing really means
What is the difference between paired end reads and overlapping reads, and then why merge overlapping reads before assembly?

[liorglic]

Just to clarify (as per the OP's question): R2 is not the reverse complement of R1. R1 and R2 come from different places in the genome, which are usually 200-500 bp apart (depending on the insert size). It is true that R1 and R2 are sequenced from different strands, but usually you don't really care about that, as all software tools which handle FastQ files will take care of this for you.