Variable amplicon size for MiSeq, bias? Maximum size?

ShellfishGene · 01-09-2014, 05:58 AM

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!

microgirl123 · 01-09-2014, 09:07 AM

The short answer to both your question is yes.

Longer amplicons tend to flop around more, interfering with other clusters. 1K is the maximum recommended length according to our Illumina FAS.

If you are mixing short and long amplicons into the same run, the smaller amplicons will preferentially bind to the flow cell. You see this in libraries with adapter dimer - the small adapter dimer binds much more easily than the larger library fragments.

saimonara · 02-12-2014, 07:47 PM

Is the maximum recommended size 1kb in order to avoid cluster overlap during bridge amplification? Have people successfully sequenced longer fragments?

The majority of my amplicons are between 600-900bp but I have some that extend to 1300bp. Will this be a problem?

Thanks!

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01-09-2014, 05:58 AM	#1
ShellfishGene Member Location: Germany Join Date: Mar 2009 Posts: 14	Variable amplicon size for MiSeq, bias? Maximum size? 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!

02-12-2014, 07:47 PM	#3
saimonara Junior Member Location: Bay Area, CA, USA Join Date: Feb 2014 Posts: 7	Is the maximum recommended size 1kb in order to avoid cluster overlap during bridge amplification? Have people successfully sequenced longer fragments? The majority of my amplicons are between 600-900bp but I have some that extend to 1300bp. Will this be a problem? Thanks! Last edited by saimonara; 02-12-2014 at 08:15 PM.

01-09-2014, 09:07 AM	#2
microgirl123 Senior Member Location: New England Join Date: Jun 2012 Posts: 200	The short answer to both your question is yes. Longer amplicons tend to flop around more, interfering with other clusters. 1K is the maximum recommended length according to our Illumina FAS. If you are mixing short and long amplicons into the same run, the smaller amplicons will preferentially bind to the flow cell. You see this in libraries with adapter dimer - the small adapter dimer binds much more easily than the larger library fragments.