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Targeted sequencing of anonymous regions
We are trying to develop a panel of anonymous regions "randomly" sampled around the genome for population genetic purposes (so we would like to be able to run this panel on 100s of samples). These would be quite small - anywhere from 100 to maybe 1200bp long.
Thus far we have been investigating PCR-based amplicon sequencing using our own set of previously developed primers in a multiplex PCR reaction. While we have reasonable coverage in our test runs on just 48 loci (~100x on a 314), most of the reads are non-specific - blasting to the taxon of interest, but not the amplicons in our panel.
Our thoughts so far are that the multiplexing is amplifying too many non-specific regions and that it might be better to submit our regions of interest to a design process.
My first question is whether a PCR-based approach (such as ampliseq) or a hybridization/capture approach (such as targetseq) will work better for our particular application.
In addition, are there any particular kits that others have had success with? Beyond the ABI kits, we are especially interested in IDTs Xgen Lockdown (as it would allow us to build our panel slowly and sequentially), and possibly in Agilent's Haloplex.
Thanks for any advice you might have.
Thus far we have been investigating PCR-based amplicon sequencing using our own set of previously developed primers in a multiplex PCR reaction. While we have reasonable coverage in our test runs on just 48 loci (~100x on a 314), most of the reads are non-specific - blasting to the taxon of interest, but not the amplicons in our panel.
Our thoughts so far are that the multiplexing is amplifying too many non-specific regions and that it might be better to submit our regions of interest to a design process.
My first question is whether a PCR-based approach (such as ampliseq) or a hybridization/capture approach (such as targetseq) will work better for our particular application.
In addition, are there any particular kits that others have had success with? Beyond the ABI kits, we are especially interested in IDTs Xgen Lockdown (as it would allow us to build our panel slowly and sequentially), and possibly in Agilent's Haloplex.
Thanks for any advice you might have.