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Old 05-11-2014, 12:30 PM   #1
Elham1
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Default Design a probe for target enrichment assay for NGS

Hi everyone,

I designed several probes for highly polymorphic region to do target enrichment assay. In particular region, I will have around 200-300 different probes that are just different in one nucleotide to distinguish different alleles. I am unsure having probes with one or two nucleotides different will be affecting the enrichment assay to run in Miseq. Can anyone help me?

Thanks.
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Old 05-11-2014, 04:19 PM   #2
nucacidhunter
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I assume probes are being used for one of in solution capture kits or a similar homebrew. The probes does not need to be exact match of target region. One or two differences in base sequences is less likely to affect your assay. The whole idea of capture is to obtain homologous region sequences which are divergent. Standard hybridisation conditions is able to capture sequences with at least 80% similarity. Altering hybridisation and washing temperature is one of the ways to change stringency. By using low stringent conditions capturing sequencing down to 70% homology is possible with the expense of increased off target capture.
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Old 05-12-2014, 02:54 PM   #3
Elham1
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Hi

Thank you for response but I actually meant that whether or not these closely matching probes adversely affect the enrichment?
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Old 05-12-2014, 04:25 PM   #4
nucacidhunter
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If they are used for in solution hybrid capture reaction to pull out homologous fragments for sequencing, closely matching probes will not have any nagative impact. Probes should be cross checked to ensure that they are not complemntary, otherwise thay will hybridise to each other not to target library fragments.
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Old 05-13-2014, 07:18 AM   #5
GW_OK
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Why not just have one (or a few) probes to the same site, if they're only differing by 1 base? This is what I think nucacidhunter was referring to.
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