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Old 04-23-2015, 12:00 AM   #1
john eimes
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Default fusion primers for Illumina Miseq

When I did 454 I just amplified my gene using fusion primers that had the gsp, then the barcode then the adaptor primer all in one oligo (barcoded forward and reverse)--and I just had IDT or whoever synthesize the oligo. I cannot seem to find out if this is doable with Illumina library prep. What is the easiest way to barcode your amplicon and prep it for Illumina sequencing? All of the instructins I have foudn seem so complicated compared to 454!

Please help, thanks
John
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Old 04-23-2015, 01:19 AM   #2
nucacidhunter
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This is done with Ilumina platform as well using similar approach. However, lack of understanding concepts such as low diversity and effect of amplicon size can result in suboptimum data. The easiest way to adopt amplicon sequencing for Illumina instruments is using two step PCR. A link for this approach is provided at post #2 in this thread: http://seqanswers.com/forums/showthr...16s#post154446

To achieve optimum diversity, 0-3 nucleotide can be added to 5’ end of primers. More info is provided in post #6 of this thread: http://seqanswers.com/forums/showthread.php?t=57796

Last edited by nucacidhunter; 04-23-2015 at 01:25 AM. Reason: Fixed link
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Old 04-23-2015, 02:24 AM   #3
john eimes
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Default lack of diversity?

Thank you for your reply. It is a targeted gene: MCH ~300 bp . I am genotyping individuals. Reason for need of NGS is high duplication resulting in many variants/individual. So, all of the amplicons are the same size, and they are plenty diverse!

What exactly does max diversity mean? What does the addition of nucleotides at the end do? Do you think I need that?

Thanks again!

John
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Old 04-23-2015, 03:20 AM   #4
nucacidhunter
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There are more info on sequence diversity in this thread: http://seqanswers.com/forums/showthread.php?t=27563

If you are going to use one primer set, then diversity will be very low. There are work arounds for sequencing low diversity libraries. For example, if sequencing output would be more than your study need, then some high diversity library can be spiked in. It is better to consult your sequencing centre before preparing your amplicon library. They can provide customised advice.
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Old 05-07-2015, 10:47 AM   #5
pmiguel
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Quote:
Originally Posted by nucacidhunter View Post
There are more info on sequence diversity in this thread: http://seqanswers.com/forums/showthread.php?t=27563

If you are going to use one primer set, then diversity will be very low. There are work arounds for sequencing low diversity libraries. For example, if sequencing output would be more than your study need, then some high diversity library can be spiked in. It is better to consult your sequencing centre before preparing your amplicon library. They can provide customised advice.
Actually, if you read all the way to the end of that thread, you will see report of the new (then) MiSeq Control Software that pretty much fixes all the issues with low bias libraries. You just need 5-10% phiX and even zero-diversity libraries produce sequence.

Only one remaining caveat -- if you really do have a stretch of zero-diversity sequence in your reads, then keep in mind that all clusters will be in a single color "channel" -- hence, effectively of a much higher effective cluster density. That is if the first base of your read is "A" for every cluster in your library, then the C, T and G channels will be empty except for a smattering of clusters from phiX library molecules. Every single other cluster will be in the "A" channel and thus this channel will be very crowded.

So you can't push the cluster density if you want to run zero-diversity libraries.

--
Phillip
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