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Hi all!
We want to sequence amplicons on the MiSeq to get (~200 bp) sequence of an exon of a specific gene. However due to variability we have to place the second PCR primer in the next exon, so one intron is included in the PCR product. As an added complication that intron is very variable in size, so the PCR products will range from ~500 bp to over 1000 bp. We want to sequence them only from one primer, as the 200 bp from that primer on are all we need. So I have two questions:
- Is there a maximum amplicon size? I remember reading someone on Sequanswers quoting tech staff with a maximum of 1000 bp, but I can't find anything official on this.
- If the long PCR products work ok, will there be a bias against them? I imagine the problem with long amplicons will be the bridge amplification. Can I expect longer amplicons do work less well in the bridge amplification, and thus generate clusters that are worse and are more likely to be excluded due to quality?
Thanks!
We want to sequence amplicons on the MiSeq to get (~200 bp) sequence of an exon of a specific gene. However due to variability we have to place the second PCR primer in the next exon, so one intron is included in the PCR product. As an added complication that intron is very variable in size, so the PCR products will range from ~500 bp to over 1000 bp. We want to sequence them only from one primer, as the 200 bp from that primer on are all we need. So I have two questions:
- Is there a maximum amplicon size? I remember reading someone on Sequanswers quoting tech staff with a maximum of 1000 bp, but I can't find anything official on this.
- If the long PCR products work ok, will there be a bias against them? I imagine the problem with long amplicons will be the bridge amplification. Can I expect longer amplicons do work less well in the bridge amplification, and thus generate clusters that are worse and are more likely to be excluded due to quality?
Thanks!
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