conceptual question about paired end sequencing
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7 months ago
nhaus ▴ 100

Imagine I am doing paired end illumina sequencing of this simple DNA.

5'-GTACGATCGTGCATCGATCGTACGTACGTACGTAGCGTACG-3'
3'-CGTACGCTACGTACGTACGTACGATCGATGCACGATCGTAC-5'


The first read would cover the bases similar to this (i.e. GTACGAT):

read 1
-------------->
5'-GTACGATCGTGCATCGATCGTACGTACGTACGTAGCGTACG-3'
3'-CGTACGCTACGTACGTACGTACGATCGATGCACGATCGTAC-5'


Then, the strand would flip over to neighboring oligos that are on the flow cell and the second read would be synthesized.

My question now is, if the second read covers the bases like this (i.e. GCATGC):

(please ignore the underscores, I don't know how to show white space in the code sample)

   read 1                             read 2
-------------->                <---------
5'-GTACGATCGTGCATCGATCGTACGTACGTACGTAGCGTACG-3'
3'-CGTACGCTACGTACGTACGTACGATCGATGCACGATCGTAC-5'


Or like this (i.e. CATGCTA):

   read 1
-------------->
5'-GTACGATCGTGCATCGATCGTACGTACGTACGTAGCGTACG-3'
3'-CGTACGCTACGTACGTACGTACGATCGATGCACGATCGTAC-5'
<-----------


I would appreciate a lot if somebody could clear this up for me.

Cheers!

sequencing illumina • 287 views
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I've cleaned up your post for you. The formatting option you were using is the block quote option, not the code formatting option. The button for the code formatting option is the 101010 button.

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7 months ago
GenoMax 108k

Thing to keep in mind is sequencing always happens in 5' --> 3' direction irrespective of the strand being sequenced.

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That makes complete sense. So its option 2. In that case I have a quick follow up question. When I align these two reads to the genome (lets say we use BWA), will read 2 be automatically converted to its reverse complement, so that it matches the direction of read 1? I am asking this, because if we download the human genome, its DNA sequence is represented as a single strand, so I guess one of the reads has to be converted.

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Aligners will consider both strands when aligning. Some aligners will allow you to restrict alignments to specific strands, if you want that.

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7 months ago
Carambakaracho ★ 2.7k

I think this is a good video to illustrate the concepts.

Disclaimer: It's an Illumina video, but I think the educational value is pretty high