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naamash 10-02-2013 04:29 AM

Defining custom index length on IEM
Hi all,

I'm sequencing 16S amplicons on MiSeq, using custom primers that have custom 12bp indexes.
How do I set up the sample sheet so that the MiSeq will perform 12 cycles of index reads? (I'm using IEM v1.6.0).


kmcarr 10-02-2013 12:51 PM

The MiSeq Control Software automatically determines how many cycles to use for the index read based on the length of the index sequences in your sample sheet.

kcchan 10-02-2013 12:54 PM

Annoyingly, IEM doesn't allow for custom indices. For that, you will have to manually generate a sample sheet. If you're unfamiliar with the layout of the sample sheet's layout, the easiest way to do this is to fill everything out in IEM with TruSeq indices and then changing the sequences to the right ones using notepad or a text editor. I don't recommend editing it in Excel as it can mess up the formatting of the sample sheet.

kmcarr 10-02-2013 01:05 PM

Technically speaking custom indexes can be used with IEM. You have to create your own custom "Assays" by adding an appropriately formatted text document to the 'SamplePrepKits' folder in the IEM program folder. You then have to add the name of your Assay to the appropriate Workflow file(s) in the 'Applications' folder, also in the IEM program directory.

If it's an assay/primer set you will be using a lot it pays to set it up this way.

kcchan 10-02-2013 04:39 PM

That's a very neat trick. This would save people a lot of time if they aren't proficient with scripting or using Excel and vlookup.

naamash 10-02-2013 10:25 PM

Thanks a lot, that's very helpful!

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