Demultiplexing MiSeq run reads.

memento · 02-06-2013, 12:38 PM

Hi,
I have added indexing sequence along the regular target (at both P5 and P7, but only P5 had unique index, low plexity 20 amplicons). How to demultiplex it outside of MiSeq control ? These are 6nt indexes...
Thanks!

kcchan · 02-06-2013, 12:55 PM

There are a few ways to do this. If you have CASAVA setup you can use it to demultiplex your MiSeq run with a modified setting to account for the inline barcodes. Alternatively, you can have the MiSeq generate a single FASTQ file with all of your data and manually demultiplex it using a tool such as fastx toolkit's barcode splitter. This method is probably the easiest if you have no experience using CASAVA.

memento · 02-06-2013, 01:03 PM

so first split - then trim right ?
thanks a bunch!

maasha · 09-10-2014, 05:58 AM

So how do you get the MiSeq to generate FASTQ files including the indices so you may demultiplex manually?

GenoMax · 09-10-2014, 06:05 AM

Do you want to do this on the MiSeq itself or do you have an offline installation of CASAVA or bcl2fastq available?

GenoMax · 09-10-2014, 06:24 AM

Best you can do on MiSeq is to generate a separate file of the tag reads. You will then need to use that for demultiplexing the data on some other machine. See post #15 for directions: http://seqanswers.com/forums/showthread.php?t=42204

With CASAVA/bcl2fastq: If you have access to the raw data folder you can run the demultiplexing using a sample sheet with dummy tags with a single sample entry (NNNN for 1D or NNNN-NNNN if 2D barcodes). This forces all reads to land in the "undetermined" pool. The tags are captured in the fastq read ID headers. You will need to do the demultiplexing yourself using this information.

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02-06-2013, 01:03 PM	#3
memento Member Location: UK Join Date: Nov 2010 Posts: 49	so first split - then trim right ? thanks a bunch! Last edited by memento; 02-06-2013 at 01:09 PM.

02-06-2013, 12:38 PM	#1
memento Member Location: UK Join Date: Nov 2010 Posts: 49	Demultiplexing MiSeq run reads. Hi, I have added indexing sequence along the regular target (at both P5 and P7, but only P5 had unique index, low plexity 20 amplicons). How to demultiplex it outside of MiSeq control ? These are 6nt indexes... Thanks!

02-06-2013, 12:55 PM	#2
kcchan Senior Member Location: USA Join Date: Jul 2012 Posts: 184	There are a few ways to do this. If you have CASAVA setup you can use it to demultiplex your MiSeq run with a modified setting to account for the inline barcodes. Alternatively, you can have the MiSeq generate a single FASTQ file with all of your data and manually demultiplex it using a tool such as fastx toolkit's barcode splitter. This method is probably the easiest if you have no experience using CASAVA.

09-10-2014, 05:58 AM	#4
maasha Senior Member Location: Denmark Join Date: Apr 2009 Posts: 153	So how do you get the MiSeq to generate FASTQ files including the indices so you may demultiplex manually?

09-10-2014, 06:05 AM	#5
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,083	Do you want to do this on the MiSeq itself or do you have an offline installation of CASAVA or bcl2fastq available?

09-10-2014, 06:24 AM	#6
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,083	Best you can do on MiSeq is to generate a separate file of the tag reads. You will then need to use that for demultiplexing the data on some other machine. See post #15 for directions: http://seqanswers.com/forums/showthread.php?t=42204 With CASAVA/bcl2fastq: If you have access to the raw data folder you can run the demultiplexing using a sample sheet with dummy tags with a single sample entry (NNNN for 1D or NNNN-NNNN if 2D barcodes). This forces all reads to land in the "undetermined" pool. The tags are captured in the fastq read ID headers. You will need to do the demultiplexing yourself using this information.