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Old 08-12-2009, 10:11 AM   #1
DPB
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Hi everyone,
I'm new to next-gen sequencing. Our library is a PCR product and I will have to design primers that include the adapter sequences. I"m still trying to get oriented and was wondering whether anyone has any into to sequencing reviews or literature (specifically Illumina/Solexa is what we're planning on using) and own primer design recommendations. (The Solexa website wasn' t specific enough and the Nature methods Jan 2008 primer was a good start, but I need a lot more detail to actually design the primers).

Thanks so much,

D
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Old 08-12-2009, 11:51 AM   #2
swbarnes2
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Hi everyone,
I'm new to next-gen sequencing. Our library is a PCR product and I will have to design primers that include the adapter sequences.
Are you sure? If your PCR primers have the adaptors, you will only get the 50 or so bases at the ends of your PCR product. And I think that'll have to include some of your primer, so you'll get even less real sequence.
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Old 08-12-2009, 12:09 PM   #3
DPB
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definitely NOT sure. what i have (or hope to have soon) is a library of 200mers cloned into a vector. what i'm trying to get is some sort of sequencing of those 200mers. i thought i was supposed to make a PCR primer that includes the adapters to amplify my 200mer from the vector. that's apparently not the case? do i use other primers to amplify from the vector and then ligate the adapters? are the sequencing primers then something i design that's at the start of my 200mer?
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Old 08-12-2009, 01:37 PM   #4
swbarnes2
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definitely NOT sure. what i have (or hope to have soon) is a library of 200mers cloned into a vector. what i'm trying to get is some sort of sequencing of those 200mers. i thought i was supposed to make a PCR primer that includes the adapters to amplify my 200mer from the vector. that's apparently not the case? do i use other primers to amplify from the vector and then ligate the adapters? are the sequencing primers then something i design that's at the start of my 200mer?
When you shear a DNA sample, the breaks happen all over the place, so the adaptors end up all over the place in the genome, so your reads start all over the genome. If you don't shear, but just put adaptors on your PCR primers, you will only sequence from the ends of your PCR. And I bet your adaptoers will have to be outside the binding part of your primer, meaning that your Illumina sequenicng will include your primer sequence. So you will get almost no insert sequence.

I once analyzed a sample that was a few hundred vectors, each with a different insert. The whole pool of plasmids was prepped like an ordinary sample, and I was able to tell the submitters "These inserts are there, with no SNPs, these inserts don't appear to be in there". Sure, most of the reads were vector, I just ignored those.

If you already have the pool of a 200-long amplicons, you probably should ligate them together, so you can shear them.
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