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Old 09-16-2011, 01:52 AM   #1
m1gre
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Location: Bristol, UK

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Default Exon duplication & deletions

Hi,

We're soon to be getting a MiSeq and one of our first projects is to look at a small number of genes for a large number of patients.

We need to be able to identify point mutations but also hoping to be able to detect whole exon duplication/deletions by measuring read depth.

Can anyone help with what would be the best library prep method for this and also what sort of read depth should we be looking at to be able to quantify our dup/dels.

Thanks,
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Old 09-16-2011, 02:50 AM   #2
ulz_peter
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are you planning to do target enrichment or amplicon sequencing?

I guess exon duplication/deletion analysis is impossible for amplicon sequencing (as you need to normalize PCR products...), unless the patient as a homozygous exon deletion (which is highly unlikely).

For target enrichment it's not entirely unfeasible but due to pre-capture, and post-capture amplification, as well as different hybridization efficiencies I don't think it is easy to correlate read-depth to number of template alleles...

However, as I never did Illumina sequencing myself, there might be other options that could work. If you've found a solution I would be happy to hear from, since we are waiting for a MiSeq as well.
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Old 09-16-2011, 02:58 AM   #3
m1gre
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Thanks for the reply,

Our plan was target enrichment as I thought this may be more feasible than the amplicon sequencing for quantitation. Was wondering if the SureSelect or Nextera might be an option. I'll contact Illumina tech support to see if they can help any further.
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Old 09-16-2011, 03:06 AM   #4
ulz_peter
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Nextera isn't a target enrichment alternative, it is an alternative way to prepare the library for sequencing (however, for Illumina I think it is now the official way, as they bought the company which produced Nextera reagents).

Sureselect should work fine, but with all the mplifications you still get the bias making it hard to correlate.
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Old 09-16-2011, 06:32 AM   #5
GW_OK
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I'd go with Illumina's new targeted cap system. It's a lot cheaper than Sureselect. As far the duplications go, were you thinking something like FPKM or something, ala RNAseq?
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