Hi everyone,
I hope you can help me, I have requested a quotation from a sequencing service company and the price is not what we expected.
We want to sequence a single gene from a large number of patients to look for mutations. The gene is less than 30kb and we are initially planning for hundreds of patients, maybe up to a thousand. The quotation was for the following set-up: sequence capture to isolate the gene of interest, followed by extensive barcoding and pooling before sequencing. Reducing the sequencing to our short gene and increasing the number of samples in each lane were considered clever ideas to minimise the price. However, the most costly part turns out to be the library prep which is done on each sample before sequence capture and any kind of pooling. Hence the hefty price.
I would be grateful if you could give me any tips on how to redesign my experiment!
My thoughts:
- Pool the samples prior to sending them in. Does anyone have experience with pooling samples before capture and sequencing? What do I have to think about when choosing how many samples to pool? The gene is only 30kb so I suppose we could pool quite a lot of samples, right?
- How many barcoded samples wold you recommend in a single sequencing lane? I have heard that 150 is feasible.
- We will buy the basic/standard bioinformatic analysis that will include SNP calling. Do you think that will be enough or will we need a skilled bioinformatician to help us out with the analysis? We have basic knowledge of bioinformatics.
- Long PCR instead of capture? But that would mean a lot of PCR runs. And I haven’t found any PCR kits that promises long enough amplicons to cover 30kb. And it doesn’t eliminate the LibPrep.
If anyone has an idea that does not involve high throughput sequencing but would give the results we want, I would be very grateful for that too.
Thank you!
I hope you can help me, I have requested a quotation from a sequencing service company and the price is not what we expected.
We want to sequence a single gene from a large number of patients to look for mutations. The gene is less than 30kb and we are initially planning for hundreds of patients, maybe up to a thousand. The quotation was for the following set-up: sequence capture to isolate the gene of interest, followed by extensive barcoding and pooling before sequencing. Reducing the sequencing to our short gene and increasing the number of samples in each lane were considered clever ideas to minimise the price. However, the most costly part turns out to be the library prep which is done on each sample before sequence capture and any kind of pooling. Hence the hefty price.
I would be grateful if you could give me any tips on how to redesign my experiment!
My thoughts:
- Pool the samples prior to sending them in. Does anyone have experience with pooling samples before capture and sequencing? What do I have to think about when choosing how many samples to pool? The gene is only 30kb so I suppose we could pool quite a lot of samples, right?
- How many barcoded samples wold you recommend in a single sequencing lane? I have heard that 150 is feasible.
- We will buy the basic/standard bioinformatic analysis that will include SNP calling. Do you think that will be enough or will we need a skilled bioinformatician to help us out with the analysis? We have basic knowledge of bioinformatics.
- Long PCR instead of capture? But that would mean a lot of PCR runs. And I haven’t found any PCR kits that promises long enough amplicons to cover 30kb. And it doesn’t eliminate the LibPrep.
If anyone has an idea that does not involve high throughput sequencing but would give the results we want, I would be very grateful for that too.
Thank you!
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