Paired End Reads Are Not Complimentary

Question

[Saket Choudhary]

I am new to NGS world. I read about paired end sequncing, however I have a few queries:

1. Is the Read Size = Fragment Size ?

2. Why are the Paired end reads not complimentary ? ( I had two fasta files and their reads are not complementary at all !)

3. When a fragment is sequnced , I need the primers too start synthesis. Lets say my "fragments" is AATGGCTTT . If I do a 2x3 bp reading would I get TTA &AAA and is this the reason for Q 2 above ?

Answer 1

[JC]

1. No. Read size is how much are you sequencing, fragment size is how large is the sequence before sequencing the borders (see http://www.illumina.com/technology/paired_end_sequencing_assay.ilmn).
2. See 1.
3. Primers are generally removed by the machine pipeline, you (unless something goes wrong) never see them in your final Fastq file.

Answer 2

[Dan D]

JC is correct. For question 2, here's some more detail. During library construction, the fragment size might be 1000 bp, to use a contrived example. Remember, that's 1000 base pairs of double-stranded DNA. If you're doing a Paired-End 100 read, that means you're starting from different ends of each strand, and would thus not expect to see any overlap in your reads. You're not sequencing the whole fragment. Does that make sense?