Whole genome sequencing primers

chrisaw01 · 11-05-2010, 11:25 AM

Hi,

I was wondering if anyone knew of a company that will generate whole genome sequencing primers for ABI/Sanger sequencing given I provide them with a whole genome sequence of a reference. I am particularly interested in this for viral (and maybe bacterial) genomes.

Thanks,
Chris

krobison · 11-05-2010, 01:08 PM

I don't offhand. It certainly is automatable.

Quick back-of-envelope calculation suggests even a Mycoplasma-class genome would be cheaper to do by SOLiD or Illumina. If you guesstimate 1 amplicon every 0.5 Kb, then a 1Mb genome is going to need 2000 amplicons. Cheap primers are $0.08/nt * 2 primers * 20 bases. So that's about $6K upfront for primers. And in the real world, some of those primer sets won't behave -- you'll either throw them out or grind out redoing them.

If you assume $1/reaction for total costs, then those 2000 amplicons will be $4000 to do double-stranded. Even at 0.25/reaction, you're getting down to what a large project could probably get per sample for SOLiD/Illumina -- some of the newer library prep kits claim $100/reaction -- throw in another $200 for library prep costs not covered. If a SOLiD 4 run can pack in 100 genomes (which would probably still give you several hundred fold coverage of each sample) for $10K, that adds a per library data acquisition cost of $100. So around $500/sample should be doable by 2nd gen.

Small viruses are probably a different story. Even there, depending on the number of samples you might be better off multiplexing libraries and pooling on a 2nd gen instrument OR doing your PCRs with barcoding primers and using a 2nd gen instrument.

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11-05-2010, 11:25 AM	#1
chrisaw01 Junior Member Location: Maryland Join Date: Oct 2010 Posts: 6	Whole genome sequencing primers Hi, I was wondering if anyone knew of a company that will generate whole genome sequencing primers for ABI/Sanger sequencing given I provide them with a whole genome sequence of a reference. I am particularly interested in this for viral (and maybe bacterial) genomes. Thanks, Chris

11-05-2010, 01:08 PM	#2
krobison Senior Member Location: Boston area Join Date: Nov 2007 Posts: 747	I don't offhand. It certainly is automatable. Quick back-of-envelope calculation suggests even a Mycoplasma-class genome would be cheaper to do by SOLiD or Illumina. If you guesstimate 1 amplicon every 0.5 Kb, then a 1Mb genome is going to need 2000 amplicons. Cheap primers are $0.08/nt * 2 primers * 20 bases. So that's about $6K upfront for primers. And in the real world, some of those primer sets won't behave -- you'll either throw them out or grind out redoing them. If you assume $1/reaction for total costs, then those 2000 amplicons will be $4000 to do double-stranded. Even at 0.25/reaction, you're getting down to what a large project could probably get per sample for SOLiD/Illumina -- some of the newer library prep kits claim $100/reaction -- throw in another $200 for library prep costs not covered. If a SOLiD 4 run can pack in 100 genomes (which would probably still give you several hundred fold coverage of each sample) for $10K, that adds a per library data acquisition cost of $100. So around $500/sample should be doable by 2nd gen. Small viruses are probably a different story. Even there, depending on the number of samples you might be better off multiplexing libraries and pooling on a 2nd gen instrument OR doing your PCRs with barcoding primers and using a 2nd gen instrument.