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constitution of a reads from single-end sequencing
Hi, everybody, I am new in the NGS field and this forum, I really have some confusing question to ask. I am sorry if I have misunderstood something.
The question is, suppose I have my library like this:
5'---adapter1.1---index---adapter1.2---27bpDNA---adapter2---PCRprimer---3'
I used Hiseq2000 single-end sequencing to sequence this library.
My opinion is the 5' of the sequence is fixed to a solid interface and sequencer use adapter2 as sequencing primer to initiate sequencing. If I set the reads length as 49 nt, then I will get exactly 49nt reads with the theoretical structure like 5'---27nt DNA---22nt adapter1.2---3'. Then the primer of adapter1.2 can be used to generate index sequence.
<1> If I am right, then the first question is why my data don't meet the above theory with a wide distribution of DNA length. (Still 27nt DNA has a large proportion)
<2> The second question is whether the complementary strand of 5'---adapter1.1---index---adapter1.2---27bpDNA---adapter2---PCRprimer---3' presents in the library and can be sequenced.
<3> There are also reads like 5'---part of adapter2---DNA---3'. I don't know where do these reads come from. Could it be caused by mixed PCR primer during sequecing?
Thanks and I am sorry if I didn't make myself clear.
The question is, suppose I have my library like this:
5'---adapter1.1---index---adapter1.2---27bpDNA---adapter2---PCRprimer---3'
I used Hiseq2000 single-end sequencing to sequence this library.
My opinion is the 5' of the sequence is fixed to a solid interface and sequencer use adapter2 as sequencing primer to initiate sequencing. If I set the reads length as 49 nt, then I will get exactly 49nt reads with the theoretical structure like 5'---27nt DNA---22nt adapter1.2---3'. Then the primer of adapter1.2 can be used to generate index sequence.
<1> If I am right, then the first question is why my data don't meet the above theory with a wide distribution of DNA length. (Still 27nt DNA has a large proportion)
<2> The second question is whether the complementary strand of 5'---adapter1.1---index---adapter1.2---27bpDNA---adapter2---PCRprimer---3' presents in the library and can be sequenced.
<3> There are also reads like 5'---part of adapter2---DNA---3'. I don't know where do these reads come from. Could it be caused by mixed PCR primer during sequecing?
Thanks and I am sorry if I didn't make myself clear.
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