I have been running the Roche HLA kits (both high and medium resolution) on a GS Junior and getting good results with the exception of one amplicon....
The A-4 amplicon is too long to be sequenced in one continuous read (~746bp).
I understand that the ends will only be sequenced in one direction but the middle section should be sequenced in both, meaning that the correct sequence can be assembled using AVA.
How can AVA be sure that the 2 sequences it uses to produce the consensus sequence are the correct ones?
If variation occurs outside of that middle region which is sequenced in both directions it cannot be verified that the start and end of the consensus sequence belong together, can it?
At the moment HLA genotyping is being done in-house (not using Conexio as Roche recommend) and because the A-4 sequences do not have MIDs on both ends of the sequence straight out of the .sff file they are being missed by the software.
Assembling the sequences seems the obvious thing to do but I'm unsure about the validity of results produced by AVA.
Anybody have any thoughts?
The A-4 amplicon is too long to be sequenced in one continuous read (~746bp).
I understand that the ends will only be sequenced in one direction but the middle section should be sequenced in both, meaning that the correct sequence can be assembled using AVA.
How can AVA be sure that the 2 sequences it uses to produce the consensus sequence are the correct ones?
If variation occurs outside of that middle region which is sequenced in both directions it cannot be verified that the start and end of the consensus sequence belong together, can it?
At the moment HLA genotyping is being done in-house (not using Conexio as Roche recommend) and because the A-4 sequences do not have MIDs on both ends of the sequence straight out of the .sff file they are being missed by the software.
Assembling the sequences seems the obvious thing to do but I'm unsure about the validity of results produced by AVA.
Anybody have any thoughts?
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