6. Frequently Asked Questions

Please refer to the AIR Technology Guide for recommendations for experimental design, sample-input amounts, and assay design.

Assay:

Q1: What is included in the AIR profiling kit?
A1: The kit includes all the necessary reagents for reverse transcription, amplification, and primers for next-generation sequencing. More details can be found, in the AIR RNA Reagents and AIR DNA Reagents Manual Information.

Q2: What is the difference between TCR/BCR CDR3 RNA kits for human and mouse?
A2: The TCR/BCR gene-specific primers for the human and mouse kits are different but all other reagents and protocols are the same.

Q3: Does the AIR profiling assay have UMI?
A3: Yes, both AIR RNA and AIR DNA profiling assays employ Unique Molecular Identifiers (UMIs) to identify and correct amplification biases and sequencing errors. The UMIs accurately quantitate the copy number of cDNA / DNA molecules used in amplification steps and are very important to detect low-abundance clonotypes above the background level.

Q4: What is the difference between AIR RNA CDR3 and Full-length Receptor Kit?
A4: The CDR3 kit amplifies approximately 250-300 bp CDR3 region, whereas the full-length kit amplifies the full variable region ~ 600 bp CDR1-CDR2-CDR3). For more information on sequencing instructions and applications for the two kits, please visit the webpage Table.

Q5: What sample types are compatible with the assay?
A5: Our assay is compatible with various sample types, including RNA or/and DNA samples isolated from whole blood, PBMC, immune cell fractions, tissue/cancer biopsies, or FFPE tissue samples. For more information on sample types and preparation guidelines, please refer to the Technology Guide; Applications of AIR profiling and visit the page for AIR RNA Sample Guidelines and AIR DNA Sample Guidelines for specific guidance for sample preparation.

Q6: What is the recommended protocol for profiling a small number of cells, such as sorted cells or fresh frozen tissue?
A6: For a small number of cells (e.g., 5,000-50,000 cells), e.g., isolated by FACS, tissue or blood microsamples, we recommend using the DirectCell Protocol for RNA DNA, which profiles directly from the cell lysate without the need to extract total RNA or DNA from the sample.

Q7: What is the optimal input amount for DNA as a starting material?
A7: To determine the optimal input amount for your samples, we recommend testing amplification efficiency using different starting amounts of DNA, e.g. 0.5 µg, 1 µg, 2 µg, 5 µg. Some samples such as FFPE blocks, or tumor biopsies contain impurities that inhibit the activity of DNA polymerase, so a maximum of 2 µg is recommended.

If you have enough input amount, we recommend running samples in triplicates, e.g. using 2×3=6 µg of DNA for each sample to get better quantitative data, as shown in Technology Guide Section 2.1, Fig 9, which shows the number of clonotypes you can obtain from different amounts of input DNA material.

Q8: What is the better choice of starting material for FFPE samples?
A8: In our experience, DNA due to its stability generally provides more robust data with FFPE samples. However, RNA can lead to a more sensitive profiling of clonotypes provided that RNA is not significantly degraded. For FFPE AIR-RNA profiling, we recommend using RNA with a RIN Score > 5, or RNA with at least 20% of fragments above 300-n. For best results, we recommend purifying both RNA and DNA from FFPE samples and running both AIR-RNA and AIR-DNA assays.

Q9: Can less than 500 ng of DNA be used for the AIR DNA Profiling Kit?
A9: As shown in Technology Guide Section 2.1, Fig 9, profiling with 500 ng or lower is possible, but the number of detected clonotypes will be reduced. Furthermore, only top abundant clonotypes (with a number of clonotype-specific cells more than 10) could be quantitated.

Sequencing:

Q10: What is the recommended number of reads for the AIR protocol?
A10: For sequencing, we recommend around 5-10 million reads per sample, depending on the number of samples in one flow cell and the required sensitivity. More information about sequencing can be found in the Manual NGS Prep and Sequencing Section.

Q11: How much PhiX is needed for the sequencing run?
A11: We recommend adding 15% of PhiX to the library (anywhere in the range 10-20% is acceptable).

Q12: Can AIR RNA and DNA samples be sequenced together in the same lane?
A12: Yes, AIR-RNA and AIR-DNA samples can be combined for NGS, but due to differences in amplicon sizes (approximately 350 bp for RNA vs. approximately 250 bp for DNA), we suggest mixing AIR-RNA with AIR-DNA at a 1.5:1 ng ratio (e.g., 150 ng of AIR-RNA with 100 ng of AIR-DNA). Ensure to use different indexing primers for RNA and DNA samples. NGS sequencing primers are the same for both AIR-RNA and AIR-DNA.

Q13: Which instruments can be used for CDR3 or full-length receptor sequencing?
A13: For most applications, we recommend using NextSeq2000 instrument with SBS-Leap sequencing chemistry kits due to low NGS cost and wide range of flow cells (between 100M to 1.8B reads for both 300-n and 600-n paired end reads). NextSeq500/550 still could be used for CDR3 profiling, but the cost of sequencing is higher than for NextSeq2000. MiSeq instrument allows sequencing for the 600 cycles (full-length receptor sequence) but its scale is limited to 5-10 samples. Our kits use the reverse complement workflow, so for MiSeq, you would need to use the protocol with one index primer and the V2/V3 Kits. To optimize your sequencing protocol please contact techsupport@illumina.com.

Q14: Can you sequence DriverMap AIR and DriverMap Human Genome Wide Expression Profiling Kits in the same flow cell?
A14: Yes, the custom sequencing primers (Reverse and Forward SeqDNA and SeqIND primers) are the same in the DriverMap AIR and DriverMap Human Genome Wide Expression Profiling Kits. The links above show the amplicon structure for both libraries. Please, note that AIR and Expression profiling samples need to be amplified with different indexing primers in order to be sequenced in the same flow cell.

Q15: Can the libraries be sequenced on MiSeq? Do you have any recommendations?
A15: Yes, the AIR library can be sequenced on MiSeq. Our kits use the reverse complement workflow, so only the V2/V3 kits are compatible with the MiSeq and you would need to use the protocol with one index primer. Attached is the Illumina Guide for Indexed Sequencing Overview. To optimize your sequencing protocol please contact techsupport@illumina.com.

Q16: Is it recommended to do sequencing on the NovaSeq 6000 or NovaSeq X Platform?
A16: As NGS with NovaSeq instruments provides a minimum of 8B reads per flow cell, NovaSeq instruments could be used for NGS analysis of AIR samples that are done on a large scale (i.e.> 500 samples).

Q17: Can sequencing be performed on NovaSeq 6000?
A17: Yes, it is possible to use two custom indexing primers with the NovaSeq 6000 Reagent Kit v1.5, as the NovaSeq 6000 machine works with a mixture of Ind1 and Ind2 sequencing primers from one well. The recommended approach is to spike Cellecta primers in the wells with Illumina primers, as described in the Spiking custom primers into the Illumina sequencing primers | Illumina Knowledge. If you prefer not to spike the primers, you can use the custom primers protocol described in the NovaSeq Series Custom Primers Guide (illumina.com).

We have a customer that used the SP flow cell and loaded 2nM with the protocol with 148-10-10 -148 cycles as follows:

Please note that Cellecta has not internally validated this protocol, to optimize your sequencing protocol please contact techsupport@illumina.com.

Q18: What are the recommended concentration and sequencing primer setup for NovaSeq 6000 kits?
A18: For NovaSeq 6000, we generally recommend starting at 100 pM (same as PhiX) if the library type loading concentration is unknown, then optimizing in 50-100 pM increments based on results. Here is Illumina resource for determining optimal loading based on PF and occupancy. To optimize your sequencing protocol please contact techsupport@illumina.com.

Q19: Can sequencing be performed on NovaSeq X?
A19: Yes, our kits use Dual DNA/RNA UDP Indexes and follow the reverse complement workflow. For loading on the NovaSeq X, follow Illumina’s protocol for NovaSeq X Custom Primers Protocol. To optimize your sequencing protocol please contact techsupport@illumina.com.

Data Analysis:

Q20: How can I analyze the data for TCR and BCR analysis?
A20: We recommend using the MiXCR software tool for alignment and AIR repertoire data analysis. Here are the options for our customers:
● Academic customers: They can use the free, user-intuitive MiXCR software available for download on Platforma.bio.
● Industry customers: MiLab offers a free trial period to test our kits on Platforma.bio or MiXCR command line version, starting with a 1-month free trial onwards. MiXCR packages include special presets for AIR kit analysis, the complete workflow can be set up with the name of the AIR Kit eg: MiXCR Guides Cellecta Kit
● Cellecta bioinformatics services: We offer bioinformatics services, including running the MiXCR pipeline and providing output reports. Custom analysis can also be performed upon request. Here is an example report: 2024-02-13 Final Report. Request a quote here.
Other bioinformatics tools/frameworks are also available to analyze immune repertoires as follows:

Tools	Published year	Aligner	Website
IMGT/HighV-QUEST	2012	GPA (NW)	https://www.imgt.org/IMGTindex/IMGTHighV-QUEST.php
IgBLAST	2013	BLAST	https://www.ncbi.nlm.nih.gov/igblast/
IMSEQ	2015	SCF matching	http://www.imtools.org/
LymAnalyzer	2015	Fast-tag	https://sourceforge.net/projects/lymanalyzer/

Q21: How do I obtain a MiXCR license?
A21: For an academic license go to https://licensing.milaboratories.com/, for a business license email licensing@milaboratories.com and provide a Cellecta kit reference.

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