# Trimming T overhang options for Illumina Stranded mRNA and Illumina Stranded Total RNA workflows

The [Illumina Stranded mRNA](https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/stranded-mrna-prep.html) and [Illumina Stranded Total RNA](https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/stranded-total-rna-prep.html?langsel=/us/) library prep workflows include the addition of a “T” nucleotide that enables ligation of the insert to the short Y adapter. The addition of the “T” nucleotide results in low base diversity for the first cycle of the forward and reverse reads, which can result in random base calls during sequencing. To mitigate the effects of a possible random base call at cycle 1, Illumina recommends trimming the first cycle nucleotide. Trimming the first cycle removes the T overhang and therefore improves subsequent analysis quality. The trimming can be done during or after FASTQ generation.

The following methods are available for trimming the T overhang. FASTQ files can be uploaded to BaseSpace Sequence Hub for processing with the recommended app. Alternatively, when running in standalone mode, use the Sample sheet method to trim the T overhang during FASTQ generation.

**BaseSpace Method**

The [FASTQ Toolkit BaseSpace app](https://basespace.illumina.com/apps/3122120/start) is used to trim the T overhang from the FASTQ files.

In the Base Trimming section, set “Trim reads at the 5’-end by n positions” to 1 to remove the first base from input FASTQ files.

**Local Run Manager**\
Enter the following two lines in the Advanced Module Settings section (Custom setting, Setting value).

* Read1StartFromCycle,2
* Read2StartFromCycle,2

**Sample Sheet Method**

Users can add the following settings to the \[Settings] section of the SampleSheet.csv file. These settings configure the FASTQ generation to start from the second cycle, skipping the T overhang.

* \[Settings]\
  Read1StartFromCycle,2\
  Read2StartFromCycle,2

**Note:** Illumina recommends running different library types in different runs. If a run does have multiple library types, one of which does not include a T overhang, these libraries must be demultiplexed separately using distinct SampleSheet.csv files because the FASTQ generation step cannot handle more than one setting per run.

**BCL Convert**

The requirements needed for trimming using BCL Convert (which is a newer version of bcl2fastq2) are different. Users can add the following settings to configure the FASTQ generation to start from the second cycle, skipping the T overhang:

* \[BCLConvert\_Settings]\
  OverrideCycles,N1Y100;I10;I10;N1Y100

Use the N1Y100 setting to skip the first cycle, but process the remaining 100 cycles of read1 and read2, given a read length of 101bp. If using alternative read lengths, for example 201bp, use the setting N1Y200;I10;I10;N1Y200. The I10 in the setting indicates a index read length of 10bp. Refer to the [BCL Convert User Guide](https://support.illumina.com/downloads/bcl-convert-user-guide.html?langsel=/us/) for more details.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #3227), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000003227%20-%20Software%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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