amplicon sequencing on MiSeq

[krobison]

I've often wondered if you couldn't include some Ns in the primer for low complexity libraries which you could then remove post sequencing along with your target primer. So your fwd primer would look like this

[P5][Seq Primer]NNNNN[Target Primer]

In theory the first 5 bases would be random which would give decent cluster identification.

There's probably some obvious reason why this wouldn't work that I haven't thought of yet.

Or maybe designing PCR's to both strands would increase the complexity enough?

A related trick I saw published was to use a variable length sequence between the Illumina primers & targeting sequence; this way the targeting sequences aren't all in phase & the complexity is significantly increased in the eye of the cluster caller.

[TonyBrooks]

I've often wondered if you couldn't include some Ns in the primer for low complexity libraries which you could then remove post sequencing along with your target primer. So your fwd primer would look like this

[P5][Seq Primer]NNNNN[Target Primer]

In theory the first 5 bases would be random which would give decent cluster identification.

There's probably some obvious reason why this wouldn't work that I haven't thought of yet.

Or maybe designing PCR's to both strands would increase the complexity enough?

[NextGenSeq]

That's a very low complexity library. You'll probably have to spike a lot of PhiX in to get good results.

[nancysch]

to start, just 4 amplicons of sizes 130-200 (without adaptors), and 32 samples.

[ECO]

How many samples and what size are your amplicons?

[nancysch]

I am a new Illumina user. What is the best way to do targeted seqeuncing (<100 amplicons) on the MiSeq?