Step by step instructions for sequencing library QC with the iSeq 100 System

Prior to sequencing on the NovaSeq 6000 instrument, the iSeq 100 system can be used for library and pooling QC. Following an iSeq 100 QC run, library pooling is rebalanced, which improves the evenness of library representation in the NovaSeq 6000 sequencing data. Use the following steps to prepare and evaluate the iSeq 100 QC run:

1. Normalize libraries for sample pooling

• Each library needs to be uniquely indexed. Quantify each library using the recommended method for the specific library type. Dilute each library to a 1 nM concentration. Note that serial dilutions provide a more accurate adjustment to the concentration.

1. Pool equal volumes of each 1 nM normalized library

• Pooling 1 µl of each 1 nM library is recommended to conserve sample volume. Using higher volumes for pooling reduces pipetting error. If sufficient volume is available for all samples for both the iSeq 100 and NovaSeq runs, the pooling volume can be increased. Note that the pooled library concentration is still 1 nM, though each individual library is diluted within the total volume of the pool.

• Example Calculation: To pool 6 libraries, add 1 µl of each 1 nM library to a 1.5 ml tube for a total volume of 6 µl, to yield a 1 nM pool. For 12 samples, there would be 12 µl of 12 uniquely indexed 1 nM libraries, or for 384 samples, there will be 384 µl of 384 pooled libraries at 1 nM.

1. Dilute the 1 nM library pool to 100 pM (or the concentration empirically optimized for the library type) with Resuspension Buffer (RSB) or 10 mM Tris-HCl, pH 8.5 for iSeq 100 loading.

• Example Calculation: The iSeq 100 Sequencing System Guide provides calculations in Sequencing Section for diluting a 1 nM library pool to 100 µl of a 100 pM library. In the above example with 6 pooled libraries, add 5 µl of the 1 nM pool to 45 µl RSB to give 50 µl of a 100 pM stock. For 12 libraries, add 10 µl of the library pool to 90 µl RSB to give 100 µl of a 100 pM pool.

1. Load 20 µl of the 100 pM pool onto the iSeq 100 and perform the run, following the steps in the iSeq 100 Sequencing System Guide

2. Run the Generate FASTQ analysis module in Local Run Manager on the iSeq 100 to enable automated demultiplexing after the run completes. The demultiplexed data are the guide for rebalancing the pool to equalize the number of reads per sample.

3. Illumina recommends to first renormalize the reads so that total % Reads Identified (PF) is 100%. This method is especially useful when making various library pooling combinations and comparing between rebalancing experiments.

• Example Calculation: Data from the Public Datasets on BaseSpace (iSeq 100: Nextera DNA Flex (Ecoli, Bcereus, Rhodobacter)) of 6 samples had 94.6% Reads Identified (PF).

• Normalize to 100% by using the following formula:

  • [(demultiplexed percentage for sample X)/(total reads identified in run (94.6 in our example))] x 100

1. This method generates the following normalized values:

2. To build the rebalanced pool for each library, multiply the initial input amount (1 µl) by the scaling factor calculated from the data above. The scaling factor is simply the ratio of the MAX (highest) Normalized % divided by the index % for the current library.

• Example Calculation: With these 6 samples, the MAX Normalized % value is 19.97.

• For this run, calculate the NDF_Bcereus_1 sample rebalance as:

  • 1 µl x (19.97/19.97) = 1 µl (where 19.97 is the max representation and 19.97 is also the sample representation as this sample was the most represented in the pool).

1. For sample NDF_Bcereus_2 we calculate:

• 1 µl x (19.97/18.42) = 1.08 µl.

1. Repeat this calculation for each of the 6 samples to see the following results:
2. The total volume of the rebalanced pool is 7.33 µl in this example. This effectively adds more volume of underrepresented samples and less volume for overrepresented samples for the final pool.

3. To verify the rebalancing of the samples, dilute this rebalanced pool to 100 pM (as in step 3 above) or modify the loading concentration if the density was not within the recommended range, and perform a second iSeq 100 run. After the rebalancing protocol is proven effective, this second iSeq 100 run may be omitted prior to sequencing on the NovaSeq. While the NovaSeq loading concentration must be determined empirically, a starting place is to load four times the concentration onto the NovaSeq. For this example run, if the iSeq 100 QC run clustered well at 100 pM, load the NovaSeq at 400 pM.

See the Sequencing Library QC with the iSeq System application note for additional information on pool rebalancing and results.

For any feedback or questions regarding this article (Illumina Knowledge Article #2698), contact Illumina Technical Support [e-mail].