7.1. Quantify and Combine Samples for NGS

In this step, the yield of products from the Index Primer PCR Reaction—the Amplified Indexed Libraries of transcripts from each sample—are measured and then pooled in equimolar amounts for sequencing.

Quantify the Amplified Indexed Libraries

1. Analyze the Amplified Indexed Libraries using one of the following methods:

• Standard Method: Separate 5 μl of Amplified Indexed Libraries on a 3% agarose-TAE gel and analyze the size distribution of NGS probes by UV transilluminator. See below for the expected results of amplified libraries generated from good-quality PBMC DNA samples. For both AIR TCR and AIR BCR assay the smear with several bright bands should be in the 200-300 bp range.

• Alternative Method: Analyze 1 µl of each of the Amplified Indexed Libraries on either an Agilent Bioanalyzer with the Agilent High Sensitivity DNA Kit (Cat.# 5067-4626) or Fragment Analyzer using the High Sensitivity NGS Analysis Kit (Cat.# DNF-473-1000) using the manufacturer’s protocol.

*Note: In some cases, excess PCR primers compromise resolution and complicate data analysis with the BioAnalyzer or Fragment Analyzer systems. For such cases, we recommend running a conventional gel to analyze the yield of amplified products.

2. Analyze yields of the Amplified Indexed Libraries. The yield should all be roughly the same for all experimental samples within +/- 2-3-fold levels and should be similar to Positive Control PBMC DNA sample. A negative control sample should not generate any significant yield of amplified products. If some samples show significantly lower yield (e.g., >5-10-fold) of amplification products, it could be an indication of differences in the amount, quality of DNA or content of lymphocyte DNA used in AIR assay. For experimental DNA samples with significantly less yield of PCR products than other samples or the Positive Control DNA sample (i.e., >5-10-fold difference), you can rerun the Amplified Indexed Libraries in the thermal cycler (see Note below) for 2-5 additional cycles. After cycling, quantify the products again relative to the Positive Control DNA. Note: Do not include the Positive Control DNA sample in additional cycles. Remove the positive control sample from the plate and keep it as a reference to assess the quantity of your PCR samples.

*Note: It is not necessary to achieve the same yield of amplified products for all samples. Adjustments for small (2-5-fold) differences in the yield between samples will be made when the samples are combined for the sequencing step following this quantification procedure.

Remove Excess PCR Primers and Combine Samples

1. Remove excess primers from the completed PCR reactions by adding 1 µl of Primer Removal Enzyme to each of the Amplified Indexed Libraries and the Negative Control sample, then incubate at 37°C for 30 minutes.

2. To ensure accurate quantification for sequencing, you need to quantitate the yield of the Amplified Indexed Libraries. The preferred method is to analyze 2 µl of each of the Amplified Indexed Libraries using either an Agilent Bioanalyzer with the Agilent High Sensitivity DNA Kit (Cat.# 5067-4626) or Fragment Analyzer using the High Sensitivity NGS Analysis Kit (Cat.# DNF-473-1000) using the manufacturer’s protocol. Quantifying the PCR products after the removal of PCR primers is more accurate than quantifying before the primer removal clean up step.

3. After primer removal and quantification, use the yield assessment of the Amplified Indexed Libraries as a basis to combine equimolar amounts of each of the Amplified Index Libraries into a single pool for NGS. For example, if the yield of Library 1 is twice that of Library 2, then mix 5 µl of Library 1 with 10 µl of Library 2. To minimize sample-to-sample sequencing variations, combine and load all experimental samples onto one NGS flow cell.

For the DriverMap AIR Assay, which generates a complex pool of amplified TCR or BCR CDR3 regions (40K-100K from 5 µg of PBMC DNA), refer to the table below for guidelines on how many samples may be combined for different instruments and read depths. Generally, you should aim for 5-10 million reads per AIR sample (starting from 5 µg of PBMC DNA) or achieve at least an average of 20 reads per DNA molecule. For Illumina instruments, AIR profiling assays require the use of 300-n paired-end reagent kits.

Instrument	Reads per flow cell	Number of samples for multiplexing per flow cell	Reads per AIR sample
NextSeq 500/550	120 million (medium throughput)	12-20	6-10M reads/sample
NextSeq 500/550	400 million (high throughput)	40-72	6-10M reads/sample
NextSeq 2000	100 million (P2 flow cell)	10-12	10M reads/sample
NextSeq 2000	400 million (P2 flow cell)	40	10M reads/sample
NextSeq 2000	1200 million (P3 flow cell)	96	10M reads/sample
NovaSeq Series	6000 million (S2)	500	10M reads/sample

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