5.3. Next-Gen Sequencing (NGS)

The Amplified NGS Index Libraries made from each total RNA sample should be run on an Illumina sequencer following the manufacturer’s instructions. Generally, we recommend setting up the sequencing reactions to generate 8-10 million reads per sample (read depth per sample). This depth works out to produce 500-1000 reads, on average, per gene—of course, low represented genes will have much fewer reads than highly represented ones.

The procedure below is based on our experience with the NextSeq 500 using the 75-cycle NextSeq 500/550 High Output Kit v2 (Illumina Cat.# FC-404-2005).

The multiplexing level may be modified to meet your experimental needs. For example, multiplex fewer samples together to generate more sequencing reads per sample (i.e., more depth) for increased and more sensitive detection of genes present across a broader dynamic range of expression levels, or, if you are mostly interested in highly expressed genes, you can sequence more samples together which will be less expensive.

Follow the standard Illumina procedures for Cluster Generation starting with 10 nM of the purified PCR sample.

1. Add 6 µl of each of the custom sequencing primers into the appropriate wells of the Illumina reagent cartridge, as follows:
   • Reverse SeqDNA NGS Primer (Read 1 Sequencing Primer) into well #20
   • Forward SeqDNA NGS Primer (Read 2 Sequencing Primer) into well #21
   • Reverse SeqIND NGS Primer (Index 1 Sequencing Primer) into well #22
   • Forward SeqIND NGS Primer (Index 2 Sequencing Primer) into well #22

Note: We do not recommend adding primers to the “Custom” wells of the reagent cartridge. We add our in-house custom sequencing primers into the Illumina premixed primer wells in order to take advantage of the PhiX internal control. Addition of our in-house sequencing primers does not affect the performance of the flow cell.

2. Perform the NGS run using 75-nt paired-end reads (or 150-nt if longer reads are desired for direct alignment to the genome) on the NextSeq, HiSeq or MiSeq. Proceed to cluster amplification using the appropriate Illumina Paired-End Cluster Generation Kit; refer to the manufacturer’s instructions for this step. The optimal seeding concentration for cluster amplification of indexed libraries is approximately 1.8 pM. Use the following program for the sequence run:
   

   Program	Custom Primer	Cycles
   Read 1:	Reverse SeqDNA	38
   Index 1:	Reverse SeqIND	6
   Index 2:	Forward SeqIND	6
   Read 2:	Forward SeqDNA	38

Note: The program has a total of 88 cycles. There are enough reagents in the 75-cycle kit (Illumina Cat.# FC-404-2005) to run at least 90 cycles.