MiSeq Index Read

PTW · 01-22-2013, 01:19 PM

Is it possible to customise the MiSeq run to do the index read after read 2 (on the read 2 strand). I've seen the Nextera protocol for dual index reads but that's not quite what I'm looking for. Thanks for any advice.

kcchan · 01-22-2013, 02:01 PM

The primers and barcodes for the index read are designed to read immediately after read 1. The primer cannot bind to the reverse complement strand during read 2. If you used your own primers and barcodes, however, you may be able to get around it, but I'm not sure what the advantage of doing that is. Reading the indices at the very end also puts you at risk of not having usable data should the run encounter a problem and stop before the index read.

PTW · 01-23-2013, 02:04 AM

Thanks. Have had to significantly customise our library due to the nature of our work so am already using custom primers and have had to move barcode from it's usual position. Getting good Read 1 and Read 2 but not index. I think I know the issue and doing the index read on the read 2 strand would likely solve it - if it is possible to programme the run to do this. If anyone has done this would be really helpful to know.

kmcarr · 01-23-2013, 10:15 AM

Quote:

Originally Posted by PTW

Is it possible to customise the MiSeq run to do the index read after read 2 (on the read 2 strand). I've seen the Nextera protocol for dual index reads but that's not quite what I'm looking for. Thanks for any advice.

Talk to your Illumina FAS or Tech Support. I don't know for sure but it may be possible to create a custom recipe for the MiSeq like you can for the HiSeq.

kcchan · 01-23-2013, 10:24 AM

Quote:

Originally Posted by PTW

Thanks. Have had to significantly customise our library due to the nature of our work so am already using custom primers and have had to move barcode from it's usual position. Getting good Read 1 and Read 2 but not index. I think I know the issue and doing the index read on the read 2 strand would likely solve it - if it is possible to programme the run to do this. If anyone has done this would be really helpful to know.

Have you considered perhaps using an inline barcode instead? This may be less of a headache than trying to hack the MiSeq recipes.

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01-22-2013, 01:19 PM	#1
PTW Junior Member Location: UK Join Date: Jan 2013 Posts: 2	MiSeq Index Read Is it possible to customise the MiSeq run to do the index read after read 2 (on the read 2 strand). I've seen the Nextera protocol for dual index reads but that's not quite what I'm looking for. Thanks for any advice.

01-22-2013, 02:01 PM	#2
kcchan Senior Member Location: USA Join Date: Jul 2012 Posts: 185	The primers and barcodes for the index read are designed to read immediately after read 1. The primer cannot bind to the reverse complement strand during read 2. If you used your own primers and barcodes, however, you may be able to get around it, but I'm not sure what the advantage of doing that is. Reading the indices at the very end also puts you at risk of not having usable data should the run encounter a problem and stop before the index read.

01-23-2013, 02:04 AM	#3
PTW Junior Member Location: UK Join Date: Jan 2013 Posts: 2	Thanks. Have had to significantly customise our library due to the nature of our work so am already using custom primers and have had to move barcode from it's usual position. Getting good Read 1 and Read 2 but not index. I think I know the issue and doing the index read on the read 2 strand would likely solve it - if it is possible to programme the run to do this. If anyone has done this would be really helpful to know.