NextGENe software's Sequence Operation Tool includes an option to Remove Duplicate Reads. This is to help improve the accuracy of coverage by condensing a family of reads from the same fragment into a single read. Many Illumina chemistries use Unique Molecular Identifiers (UMIs) to label reads in order to help with this process. These UMIs could be in the i7 and/or i5 indexes. And NextGENe software's Process UMIs based on I2 Index Files to remove duplicates from the R1 and R2 files.
NextGENe requires that the UMIs be in the I2 index file to utilize the Process UMIs based on I2 Index Files function. It is likely that the Index files are not generated alongside the R1/R2 fastq files by default. These are steps to assist with generating the Illumina index file that includes the UMIs.
Generate Index Files
Update Sample Sheet
Here are suggestions for V2 sample sheet.
Configure the [Reads] section to include the appropriate lengths for Index1Cycles and Index2Cycles.
Configure the [BCLConvert_Settings] section:
OverrideCyclesneeds to be appropriately configured as to prevent UMIs for being trimmed- Set the
CreateFastqForIndexReadsparameter to 1 TrimUMIneeds to be set to 0
Use bclconvert to create the index fastq files
Remove Duplicate Reads utilizing UMIs in Index File
If the UMIs are in the I2 file, then simply load the I2/R1/R2 fastq.gz files into the Sequence Operations tool. If the UMIs are in the I1 file, rename I1 to I2. Then load the new I2 with the R1/R2 files into the Sequence Operations tool.
Open NextGENe -> Tools -> Sequence Operations and select the Remove duplicate reads button.
Select Process UMIs based on I2 Index File, update settings as desired, and click OK.
The Output will include Processed and Removed fastq files as well as a log showing the count of duplicates. The Processed fastq files can now be used to show one read per UMI family.