Dear All,
I would like to ask for some help with design of a sequencing project. The aim would be to sequence the exons of 20 genes in 1000 subjects. I've look into the forum and did see some threads that have this or that bit of information, but none of them give a clear pipeline or design of a similar project. I do apologize for being very basic, though.
So far, this is what I've got - I am planning on pooling 20 subjects and do a PCR amplification of the exons, then combine the PCR products and then multiplex sequence them on Illumina HiSeq 2000. This is a very broad outline and I've got no idea about the details. I assume I first need to design primers to capture all of the exons, but what size my products should be?
For example, I would need say 350 products to capture all of the exons, then I would run 350 x 50 reactions to get the products (50 reactions would be the 1000 subjects divided by 20 because of the pooling). So far so good... then what do I do? my understanding is that I can pool again the PCR products - so I can combine all the products (all 350) of one 20 subjects' pool and then label it for sequencing? so, there will be 50 sequencing reads?
again, I am sorry if this is very basic, but I've got limited options of support and got really confused at the moment...
many thanks for your time and kind help!
I would like to ask for some help with design of a sequencing project. The aim would be to sequence the exons of 20 genes in 1000 subjects. I've look into the forum and did see some threads that have this or that bit of information, but none of them give a clear pipeline or design of a similar project. I do apologize for being very basic, though.
So far, this is what I've got - I am planning on pooling 20 subjects and do a PCR amplification of the exons, then combine the PCR products and then multiplex sequence them on Illumina HiSeq 2000. This is a very broad outline and I've got no idea about the details. I assume I first need to design primers to capture all of the exons, but what size my products should be?
For example, I would need say 350 products to capture all of the exons, then I would run 350 x 50 reactions to get the products (50 reactions would be the 1000 subjects divided by 20 because of the pooling). So far so good... then what do I do? my understanding is that I can pool again the PCR products - so I can combine all the products (all 350) of one 20 subjects' pool and then label it for sequencing? so, there will be 50 sequencing reads?
again, I am sorry if this is very basic, but I've got limited options of support and got really confused at the moment...
many thanks for your time and kind help!
Comment