6.3. Next Generation Sequencing

The Amplified 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).

*Note: The procedure below is based on our experience with the NextSeq 500/550 using the 150-cycle NextSeq 500/550 Mid Output Kit v2.5 (Illumina Cat.# 20024909) and NextSeq 2000 using 100-cycle P2 flow cell with XLeap-SBS chemistry (Illumina Cat.# 20100987).

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 low abundant transcripts (e.g. cytokines) present across a broader dynamic range of abundance/expression levels, or, if you are mostly interested only in highly abundant/expressed transcripts, you can sequence more samples together which will be less expensive.

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

NextSeq 500/550
For the NextSeq 500/550, 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 NextSeq 500/550 reagent cartridge. We add our in-house custom sequencing primers into the Illumina premixed primer wells to take advantage of the PhiX internal control. The addition of our in-house sequencing primers does not affect the performance of the flow cell.

NextSeq 2000
For the NextSeq 2000, you must use the Custom wells of the reagent cartridge. HT1 buffer is required to mix with custom sequencing primers. 1.8 µl of each of the Read primers are mixed with 600 µl of HT1 buffer and loaded into Custom 1 well. 3.6 µl of each of the Index primers are mixed with 600 µl of HT1 buffer and loaded into the Custom 2 well.

• Forward SeqDNA NGS Primer (Read 1 Sequencing Primer) and
• Reverse SeqDNA NGS Primer (Read 2 Sequencing Primer) into Custom 1 well
• Forward SeqIND NGS Primer (Index 1 Sequencing Primer) and
• Reverse SeqIND NGS Primer (Index 2 Sequencing Primer) into Custom 2 well
  

2. Perform the NGS run using 150-cycle Paired End reads on the NextSeq 500/550 or 100-cycle Paired End reads on the NextSeq 2000 instrument. The optimal loading concentration for cluster generation of indexed libraries is approximately 1.8 pM for NextSeq 500/550 and 800pM for NextSeq 2000. Use the following program for the sequence run, and for the NextSeq 2000 be sure to also indicate the custom wells:

NextSeq 500/550

Program	Custom Primer	Cycles
Read 1:	Forward SeqDNA	70
Index 1:	Forward SeqIND	10
Index 2:	Reverse SeqIND	10
Read 2:	Reverse SeqDNA	70

*Note: The program has a total of 160 cycles. There are enough reagents in the 150-cycle kit (Illumina Cat.# 20024909) to run at least 160 cycles.

NextSeq 2000

Program	Custom Primer	Cycles	Custom Well
Read 1:	Forward SeqDNA	59	Custom 1
Index 1:	Forward SeqIND	10	Custom 2
Index 2:	Reverse SeqIND	10	Custom 2
Read 2:	Reverse SeqDNA	59	Custom 1

*Note: The program has a total of 138 cycles. There are enough reagents in the 100-cycle P2 flow cell (Illumina Cat.# 20100987) to run at least 138 cycles.