Hi guys
What do you think the best way to sequence a 12kb gene in multiple individuals is? Conventional sanger costs are still expensive, advances in high throughput sequencing is more attractive, but which platform?
I have been looking at Illumina TruSeq library prep with GAII sequencing, I would have to multiplex 96 samples on a flowcell, 12 samples per pool, and do single end 36bp reads, allowing 6bp for index barcode.
I have also been looking at the protocol in this paper:
which uses position specific barcodes per 96 well plate, pooling and then ligation of a plate specific tag - what are people's thoughts on this? There will be a massive number of PCR reactions as I would need to have approx 27 overlapping fragments of the gene per individual to PCR, followed by addition of the plate specific tag through ligation in library prep step and then sequencing.
thoughts are welcome.
Vanisha
What do you think the best way to sequence a 12kb gene in multiple individuals is? Conventional sanger costs are still expensive, advances in high throughput sequencing is more attractive, but which platform?
I have been looking at Illumina TruSeq library prep with GAII sequencing, I would have to multiplex 96 samples on a flowcell, 12 samples per pool, and do single end 36bp reads, allowing 6bp for index barcode.
I have also been looking at the protocol in this paper:
which uses position specific barcodes per 96 well plate, pooling and then ligation of a plate specific tag - what are people's thoughts on this? There will be a massive number of PCR reactions as I would need to have approx 27 overlapping fragments of the gene per individual to PCR, followed by addition of the plate specific tag through ligation in library prep step and then sequencing.
thoughts are welcome.
Vanisha
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