[bioinfosm]

This means you cannot "re-use" a flow cell. that would have run just a few lanes?

From what I know ... you waste the lanes that were left empty. no going back to use them later

[sci_guy]

DNAcowboy, if your main application is sequencing 1 - 10 genes in multiple patients then traditional Sanger sequencing would be best, unless you're wishing to sequence the same genes in scores of people, then a custom resequencing array would be more advisable. But, if you wish to do deep sequencing of tumour DNA for rare somatic mutations in your genes of interest then the read depth of next-gen seq is needed.

Applied biosystems has the VariantSeqR primer library - a good place start with primer design. If your gene of interest is reasonably sized it will probably take about 20 - 25 primer pairs to amplify the exons and 1kb upstream of TSS. For 10 genes lets say: 200 - 250 reactions/person. A liquid handler robot and Invitrogen E-gels will make your life easy here. If you do the math on PCR/Seq costs it will probably be somewhat cheaper than a 454/Solexa lane plus you can buy very mature and nice GUI based software to do your analysis (e.g Sequencher). It is still the Wild West when it comes on software pipelines with next-gen. If you are a Linux shell geek then you'll be fine there.

If you're sequencing a large pool of people the hefty Affymetrix resequencing array design cost and minimum chip purchase requirements is a non-issue and this option is worth looking into.

Next-gen seq of 1 - 10 genes would require up-front selection of your DNA from the genome, so you'll need short PCR exon amplification, LR-PCR tiling or Nimblegen chip-seq as a "front-end"... a cost of a few thousand per person here (unless you recycle your Nimblegen chips and buy a Maui hyb station, itself a $20K piece of equipment).

The viral genome sequencing application seems built for next-gen sequencing. If these jobs are few and far between then next-gen service sequencing would be a good option.

Hope this helps.

[DNAcowboy]

It sure does help, thanxx.

People around me, for sure are working on a small number of genes but you'll find funny how this number exponentially increases when you mention the nextgen sequencers capacity. We indeed work on rare mutations for various diseases and time has come to go further, over the well known mutations.

We also are onto small viruses genomes and that's part of the reason I would like to understand the math of running more than 1 sample on a NGS.

I actually own a 8 capillary-CEquencer and the math I have for 1Mb @ $20K should be indeed less on a 96 capillary system (with ABI chemistry) but presumably still over $10K. Will you agree on that kind of cost for home-CEquencing?

[sci_guy]

I actually own a 8 capillary-CEquencer and the math I have for 1Mb @ $20K should be indeed less on a 96 capillary system (with ABI chemistry) but presumably still over $10K. Will you agree on that kind of cost for home-CEquencing?

Don't know about CEquencing pricing. The pricing I had calculated was for sending purified DNA in 96 well plates to a ABI 3730xl service sequencing centre.

If I plug your numbers into my budgeting spreadsheet I get:
Cost for one person $5K/person
Cost for ten people $2.5K/person

Costs include oligos, PCR, PCR cleanup and service sequencing. The reason for the large price drop per person when going from 1 to 10 people sequenced is recycling PCR oligos. BTW, Invitrogen offers oligo synthesis of full 96-well plates at a VERY competitive price.

If you have access to Affymetrix array hardware their resequencing chips are good in terms of value and throughput if you have numbers of patients and numbers of bases to sequence that much their wafer size. In the table below Array Format translates to the number of arrays etched into one wafer of silicon and Maximum Capacity is the max number of bases sequenced in both directions on dsDNA. Format-49 costs are a once only free of around $24K for chip design then $600 odd per chip.

Array Format Maximum Capacity
49 303,366
100 117,254
169 47,974

If you use next-gen-seq you will need to price in the "front-end" enrichment technologies (LR-PCR/PCR/Chip-Seq). This can really change the equation.

We also are onto small viruses genomes and that's part of the reason I would like to understand the math of running more than 1 sample on a NGS.

Read some webinar presentations on the Illumina website. Good place to start.

[horigen]

The providers I talked to (GATC Bio, SeqWright, Prognosys, BCGSC, Illumina...couple others) didn't mention additional control costs.

BCGSC was cheapest (still close to $4k USD IIRC), however my company didn't want to sign over all rights to the data, which was kind of a weird requirement for a service facility.

This site quotes a price of "about 800 dollars/lane for 18 cycles to 1,200 dollars for 60 cycles/lane".

http://www.genomecenter.ucdavis.edu/dna_technologies/uhtsequencing.html