7.3. Next Generation Sequencing Full-length region

Library QC

1. Measure the concentration of the library using using Qubit HSDNA and Agilent Tapestation 4200 HSDNA1000.

2. Convert to molar concentration using the formula below:

3. Dilute your library to 800pM for loading concentration (larger fragments) using RSB to final volume 24 μl and add 5-10% PhiX Control.

4. Example concentration (used for final loading):

Library Pool ID	Concentration (mass)	Fragment size	Concentration (nm)
Pool1	1.615	536	4.5
Pool2	1.15	375	4.6
Pool3	0.58	393	2.3
Pool4	1.18	332	5.4

Sequencing on NextSeq 1000/2000

The instructions below are for full-length receptor region sequencing (~500 nt) using the NextSeq 1000/2000 P2 300M Reagents (600 Cycles) catalog number 20075295.

*For full-length sequencing, a 600-cycle kit is required which is available only on the NextSeq 1000/2000 P1 Reagents, P2 Reagents, or MiSeq v3 Reagent Kit. If using our custom primers with PhiX, you only need to buy the Illumina Read Primer Set Kit (Catalog Number 20112859).

1. Custom primers are prepared using HT1, and then added to the custom wells in the NextSeq 1000/2000 reagent cartridge. Make sure that the reagent cartridge is thawed and inspected before proceeding.
• Use HT1 to dilute each custom read primer mix to yield 600 µl at 0.3 µM final concentration.
• Use HT1 to dilute each custom index primer mix to yield 600 µl at 0.6 µM final concentration.

2. When using custom or third-party libraries alongside PhiX or Illumina libraries, prepare your custom read primers by spiking into HP21 and custom index primers by spiking into BP14. Kit Name Catalog # NextSeq 1000/2000 Read & Index Primers 20046115.
• Add each custom read primer mix to 600 μl HP21 for a 0.3 μM final concentration.
• Add each custom index primer mix to 600 μl BP14 for a 0.6 μM final concentration

3. Before loading the sample and custom primer, invert the cartridge several times to ensure complete reagent mixing. You will not be able to do this once everything is loaded.

4. Transfer 600 μl of the custom read primer into “Custom 1” well and 600 μl of the custom index primer into “Custom 2”. You can use either well, but it is helpful to have a fixed well for repeated use.

5. When in manual setup mode, after selecting your read lengths, set the custom primer drop-down under each read to the correct well.

Program	Cycles
Custom 1:	308
Custom 2:	10
Custom 2:	10
Custom 1:	308

6. This will direct the instrument to pull the custom primer from these wells, not the stock material inside the cart. Once that is complete, you can follow the standard protocol to start the run.

*Note: The program has a total of 636 cycles. There are enough reagents in the 600-cycle kit to run at least 636 cycles.

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