Pool and Denature Libraries
Sequencing System: NovaSeq X, standard
Protocol A
Use this protocol to denature and dilute libraries that have been normalized using standard library quantification and quality control procedures recommended in the library prep documentation. The NovaSeq X Series 10B Flow Cell and the NovaSeq X Series 1.5B Flow Cell have the same recommendations per lane. However, the NovaSeq X Series 10B Flow Cell has eight lanes and the NovaSeq X Series 1.5B Flow Cell has two lanes.
To obtain the optimal seeding concentration to yield the best %PF, you might need to perform a titration of your library type.
The following consumables are required to denature and dilute libraries.
|
Consumables |
Supplier |
Purpose |
|---|---|---|
|
1 N NaOH |
General lab supplier |
Diluting to 0.2 N for denaturing libraries. |
|
Microcentrifuge tube, 1.5 ml |
VWR, catalog # 20170-038, or equivalent |
Combining volumes |
|
Pipette tips, 20 μl |
General lab supplier |
Pipetting reagents and libraries. |
|
Pipette tips, 200 μl |
General lab supplier |
Pipetting reagents and libraries. |
|
Pipette tips, 1000 μl |
General lab supplier |
Pipetting reagents and libraries. |
|
Pre-load buffer |
Illumina, provided in the NovaSeq X Series reagent kit |
Neutralizing denatured libraries. |
|
Resuspension Buffer (RSB) |
General lab supplier |
Diluting libraries to the required loading concentration. |
|
Water, laboratory-grade |
General lab supplier |
Diluting NaOH for denaturing libraries. |
|
[Optional] PhiX Control v3 |
Illumina, catalog # FC-110-3001 |
Spiking in PhiX control. |
Prepare NaOH
Prepare a fresh dilution of 0.2 N NaOH to denature libraries for sequencing. Extra volume is prepared to prevent small pipetting errors from affecting the final NaOH concentration.
| 1. | [25B] Combine the following volumes in a microcentrifuge tube. |
|
Reagent |
Volume for One 25B Flow Cell (µl) |
Volume for Two 25B Flow Cells (µl) |
|---|---|---|
|
Laboratory-grade water |
120 |
240 |
|
Stock 1 N NaOH |
30 |
60 |
These volumes result in 150 µl 0.2 N NaOH for one flow cell or 300 µl 0.2 N NaOH for two flow cells.
| 2. | [10B] Combine the following volumes in a microcentrifuge tube. |
|
Reagent |
Volume for One 10B Flow Cell (µl) |
Volume for Two 10B Flow Cells (µl) |
|---|---|---|
|
Laboratory-grade water |
80 |
160 |
|
Stock 1 N NaOH |
20 |
40 |
These volumes result in 100 µl 0.2 N NaOH for one flow cell or 200 µl 0.2 N NaOH for two flow cells.
| 3. | [1.5B] Combine the following volumes in a microcentrifuge tube. |
|
Reagent |
Volume for One 1.5B Flow Cell (µl) |
Volume for Two 1.5B Flow Cells (µl) |
|---|---|---|
|
Laboratory-grade water |
20 |
40 |
|
Stock 1 N NaOH |
5 |
10 |
These volumes result in 25 µl 0.2 N NaOH for one flow cell or 50 µl 0.2 N NaOH for two flow cells.
| 4. | Vortex, and then centrifuge the tube to mix. |
Dilute Libraries and Add PhiX Control
| 1. | Dilute libraries to 2 nM using RSB. |
| 2. | [25B] In a new 1.5 ml microcentrifuge tube, dilute libraries to the desired final loading concentration using RSB. |
The final volume should be 56 µl per sample well.
|
Final Loading Concentration (pM) |
2 nM Library (µl) |
RSB (µl) |
|---|---|---|
|
90 |
12.6 |
43.4 |
|
100 |
14.0 |
42.0 |
|
110 |
15.4 |
40.6 |
|
120 |
16.8 |
39.2 |
|
130 |
18.2 |
37.8 |
|
140 |
19.6 |
36.4 |
|
150 |
21.0 |
35.0 |
|
160 |
22.4 |
33.6 |
|
170 |
23.8 |
32.2 |
|
180 |
25.2 |
30.8 |
| 3. | [10B, 1.5B] In a new 1.5 ml microcentrifuge tube, dilute libraries to the desired final loading concentration using RSB. |
The final volume should be 34 µl per sample well.
|
Final Loading Concentration (pM) |
2 nM Library (µl) |
RSB (µl) |
|---|---|---|
|
90 |
7.7 |
26.3 |
|
100 |
8.5 |
25.5 |
|
110 |
9.4 |
24.6 |
|
120 |
10.2 |
23.8 |
|
130 |
11.1 |
22.9 |
|
140 |
11.9 |
22.1 |
|
150 |
12.8 |
21.2 |
|
160 |
13.6 |
20.4 |
|
170 |
14.5 |
19.5 |
|
180 |
15.3 |
18.7 |
| 4. | [Optional] Spike in 1–2% nondenatured PhiX as follows. |
When spiking in PhiX, 1% is the recommended amount for well balanced libraries. Low-diversity libraries can require more. To use a PhiX control with low-diversity libraries, contact Illumina Technical Support for guidance.
| a. | Quantify PhiX. |
| b. | Dilute 10 nM PhiX to 300 pM using RSB. |
| c. | Add diluted PhiX to nondenatured library. |
|
Flow Cell Type |
PhiX (µl) |
Nondenatured Library (µl) |
|---|---|---|
|
25B |
1.6 |
56 |
|
10B or 1.5B |
1 |
34 |
Denature Libraries
| 1. | Add 0.2 N NaOH to the nondenatured library tube and optional PhiX. |
|
Flow Cell Type |
0.2 N NaOH (µl) |
|---|---|
|
25B |
14 |
|
10B or 1.5B |
8.5 |
| 2. | Cap and then vortex briefly. |
| 3. | Incubate at room temperature for 5 minutes to denature. |
| 4. | Add pre-load buffer to neutralize. |
|
Flow Cell Type |
Pre-Load Buffer (µl) |
|---|---|
|
25B |
210 |
|
10B or 1.5B |
127.5 |
| 5. | Cap and then vortex briefly. |
| 6. | Centrifuge at 280 × g for up to 1 minute. |
| 7. | Store libraries on ice until transferred to the library tube strip. |
After denaturing and diluting the libraries, you are ready to load the lyo insert and library tube strip into the reagent cartridge in preparation for sequencing. Refer to the sequencing system product documentation for more information.
Revision History - Protocol for Standard Loading
|
Document |
Date |
Description of Change |
|---|---|---|
|
Document # 200047034 v00 |
June 2024 |
Initial release. |