7.3. Next Generation Sequencing

The Amplified AIR 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 5-10 million reads per sample (read depth per sample). This depth works out to produce 20-50 reads, on average, per UMI.

*Note: The procedure below is based on our experience with the NextSeq 500/550 using the 300-cycle NextSeq 500/550 High Output Kit v2.5 (Illumina Cat.# 20024908). For other Illumina instruments, please contact Illumina Tech Support Team for optimal NGS instructions.

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 clonotypes present across a broader dynamic range of abundance/expression levels, or, if you are mostly interested only in highly abundant/expressed clonotypes, 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. We recommend adding 15% of PhiX to the library.

1. Add 6 µl of each of the custom sequencing primers into the appropriate wells of the Illumina reagent cartridge, as follows:
    
   • Forward SeqDNA NGS Primer (Read 1 Sequencing Primer) into well #20
   • Reverse SeqDNA NGS Primer (Read 2 Sequencing Primer) into well #21
   • Forward SeqIND NGS Primer (Index 1 Sequencing Primer) into well #22
   • Reverse 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 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 300-nt paired-end reads on the NextSeq. 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 for NextSeq500/550. Use the following program for the sequence run:
    
   

   Program	Custom Primer	Cycles
   Read 1:	Forward SeqDNA	148
   Index 1:	Forward SeqIND	10
   Index 2:	Reverse SeqIND	10
   Read 2:	Reverse SeqDNA	148

*Note: The program has a total of 316 cycles. There are enough reagents in the 300-cycle kit (Illumina Cat.# 20024908) to run at least 316 cycles.