Hi,
I'd love to receive some advice on a strategy to sequence PCR amplicons on a MiSeq.
I'd like to simultaneously sequence ±300 PCR products from control and 'experimental' samples on a MiSeq. The PCR products will be ±200-400bp long.
For this, I'll design ±300 primerpairs to amplify the regions of interest. Then, I'd like to sequence these simultaneously. What would be the best way to go
1) Perform 'regular' PCR with gene-specific PCR primers and pool 'control' PCR products and 'experimental' products on two populations. Then ligate on a barcoded adapter for PE sequencing? Pool and sequence? What kit would work best for this?
2) Would it be easier to add a short linker to the primers and use these as priming sites in a next PCR reaction in which barcoded adapters are added to the pooled PCR products? If so, how long should this adapter be? And did anyone try this method before (what sequence would you recommend)?
How problematic is it when during sequencing all clusters give the same nt sequence (low complexity at the same region in the DNA). The entire chip may light up, does this make it hard to base-call? What are the solutions? I only have two pooled samples (control vs experimental), could I use a mix of barcodes for each? Or should I mix in the PhiX library? At what ration?
I'd prefer the first option, since it's less work. I'm just not sure how efficient the adapter annealing is in the kits.
Any help/advice would be much appreciated.
cheers
I'd love to receive some advice on a strategy to sequence PCR amplicons on a MiSeq.
I'd like to simultaneously sequence ±300 PCR products from control and 'experimental' samples on a MiSeq. The PCR products will be ±200-400bp long.
For this, I'll design ±300 primerpairs to amplify the regions of interest. Then, I'd like to sequence these simultaneously. What would be the best way to go
1) Perform 'regular' PCR with gene-specific PCR primers and pool 'control' PCR products and 'experimental' products on two populations. Then ligate on a barcoded adapter for PE sequencing? Pool and sequence? What kit would work best for this?
2) Would it be easier to add a short linker to the primers and use these as priming sites in a next PCR reaction in which barcoded adapters are added to the pooled PCR products? If so, how long should this adapter be? And did anyone try this method before (what sequence would you recommend)?
How problematic is it when during sequencing all clusters give the same nt sequence (low complexity at the same region in the DNA). The entire chip may light up, does this make it hard to base-call? What are the solutions? I only have two pooled samples (control vs experimental), could I use a mix of barcodes for each? Or should I mix in the PhiX library? At what ration?
I'd prefer the first option, since it's less work. I'm just not sure how efficient the adapter annealing is in the kits.
Any help/advice would be much appreciated.
cheers
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