How to Install Bioo Scientific’s NEXTflex Barcode Indices for Illumina Experiment Manager

Instructions for installing NEXTflex adapter indeces in Illumina Experiment Manager

Introduction

Illumina Experiment Manager (IEM) is a user interface for assembling input CSV (comma-separated variables) data that the sequencer software can read directly. The information included has reagent barcode reference, run name, investigator name, sample names, read adapter sequences, indices used and other tracking information for the pooled samples. Since Bioo Scientific’s indices are not included by default with IEM, specific index information needs to be added to the IEM data source.

 

Two approaches

In order to incorporate NEXTflex indices, two methods are available:

Directly modify the resulting CSV file with corrected indices. All files generated by IEM can be opened in any text editor or Microsoft Excel after generation. At that point the user can directly modify indices (or any other element of the file) prior to loading into the Illumina sequencing software. While this may be the quickest option for a one-off sequencing run, we highly recommend setting up IEM with NEXTflex indices (method 2) so human input errors are minimized.

Set up custom SamplePrepKit files so indices are read directly by IEM. The remainder of this guide concerns setting up NEXTflex data files for repeated usability. To generate a sample sheet with more than 96 samples, see our guide for making this process faster and easier, after the following steps have been performed. 

 

How to set up a SamplePrepKit file

IEM stores barcode sets as text files with a specific format. These barcode sets are then plugged into the various Applications listed in IEM. These barcode sets are labeled as SamplePrepKits.

Bioo Scientific has already generated the appropriate data files for the following NEXTflex Kits, which can be downloaded from the table below.

SamplePrepKit File Name NEXTflex Barcode Kits Covered
NEXTflex 6nt Barcodes.txt NEXTflex DNA Barcodes
NEXTflex ChIP-Seq Barcodes
NEXTflex PCR-Free Barcodes
NEXTflex RNA-Seq Barcodes
NEXTflex Bisulfite-Seq Barcodes*

*Note: selected indices from full set 
NEXTflex 8nt Barcodes.txt  NEXTflex-96 DNA Barcodes
NEXTflex-96 ChIP-Seq Barcodes
NEXTflex-96 RNA-Seq Barcodes 
NEXTflex Dual Index Barcodes.txt
NEXTflex Dual-Indexed DNA Barcodes 
NEXTflex HT Barcodes.txt NEXTflex-HT DNA Barcodes 
NEXTflex 16S V4 12nt Barcodes.txt  NEXTflex 16S V4 Amplicon-Seq Kits 
NEXTflex 12nt Amplicon Barcodes txt. NEXTflex 16S V1 – V3 Amplicon-Seq Kits
NEXTflex 16s V4 Amplicon-Seq 2.0 Kits
NEXTflex 18S ITS Amplicon-Seq Kits
NEXTflex Amplicon Panels
NEXTflex qRNA 8nt Barcodes.txt NEXTflex qRNA-Seq Kit  v2
NEXTflex Rapid Directional qRNA-Seq Kit
 NEXTflex Small RNA 6nt Barcodes.txt NEXTflex Small RNA-Seq Kit v2
NEXTflex Small RNA-Seq Kit v3 

 

To install the above SamplePrepKit files, first locate the installation path for IEM. The default installation at time of writing on Windows 7 is:

C:\Program Files (x86)\Illumina\Illumina Experiment Manager

Within the Illumina Experiment Manager folder, navigate to the SamplePrepKits folder. Inside this folder are a number of pre-installed Illumina barcode sets.

Copy the appropriate downloaded NEXTflex Barcodes.txt file into the SamplePrepKits directory. The barcode set is now placed so IEM can read its contents.

 

Plug-in the NEXTflex Barcode sets into IEM Applications

IEM requires that barcode sets are registered with each applicable application. Navigate back to the main directory and open the Applications folder. This folder should have a similar path to the below:

C:\Program Files (x86)\Illumina\Illumina Experiment Manager\Applications

The applications folder features separate sequencing build text files that provide specific settings. Amplicon pools may be handled differently from Resequencing, for example.

To incorporate the Bioo Scientific NEXTflex Barcode sets into a specific application, open the chosen application text file using a text editor such as Notepad on Windows. You may need to open Notepad or other editors as Administrator in Windows 7 as IEM places write protections on its files.

For example, I want to update ChIP-Seq.txt with Bioo Scientific’s 6nt Barcodes. The file appears in Notepad below:

Adding NEXTflex barcode indeces to Illumina Experiment Manager

To add the Bioo Scientific 6nt barcode set to the ChIP-Seq application, find the

[Compatible Sample Prep Kits]

line and paste the following underneath (in addition to other Sample Prep Kits mentioned):

Bioo NEXTflex 6nt Barcodes

(For the 8nt Barcodes, paste Bioo NEXTflex 8nt Barcodes)

Below is the file after editing:

Imposting NEXTflex adapter indeces to Illumina Experiment Manager

 

Save the file. If Notepad displays an Access Denied warning, reopen Notepad under Administrator mode and repeat the preceding steps.

Correcting Access Denied warning in Illumina Experiment Manager

 

 

Open IEM and Create a Sample Sheet

Now that the NEXTflex Barcode indices are properly set up, proceed with generating a new sample sheet for the next sequencing run as usual. When the Workflow Parameter screen appears, the Bioo NEXTflex adapters will be selectable in the barcode set dropdown menu: 

Creating a sample sheet in Illumina Experiment Manager

Moving to the Sample Selection spreadsheet, the Index1 (I7) column displays B001 through B048 (C001 to C096 for the 8 nt Barcode set), which correspond to Barcodes 1 to 48 of the NEXTflex 6 nt Barcode kits. At this point, the proper barcode indices have been implemented in IEM and are ready for repeated use. For generating a sample sheet with more than 96 samples, see our guide How to Easily Generate Sample Sheets with More than 96 Samples in Illumina Experiment Manager.

If you have any questions, please contact Bioo Scientific at 1-888-208-2246, 1-512-707-8993 or by email at BiooNGS@biooscientific.com.