Trim T-Overhang (Optional)

This step trims the first cycle nucleotide at FASTQ generation, removing the T-overhang. Refer to NextSeq 1000/2000 Product Documentation for more information on dark cycle sequencing.

Illumina Stranded mRNA Prep adds a thymine (T) nucleotide to cDNA inserts, which enables ligation of the insert to the adapter. The addition of the T nucleotide creates low diversity in the first cycle of each insert read. Low diversity can result in random base calls at the first sequencing cycle. To improve analysis quality, Illumina recommends trimming the first cycle nucleotide.

1.

To trim using the FASTQ Toolkit app on BaseSpace Sequence Hub, perform the following actions.

a.

Expand the base trimming settings section.

b.

Enter 1 in the Trim reads at the 5ʹ-end by n positions field.

2.

To trim using a sample sheet, add the following settings to the sample sheet file.

•

Read1StartFromCycle,2

•

Read2StartFromCycle,2