Table 1.
Summary of antibody gene sequencing using NAb-seq.
| Cell type | Hybridoma |
Single B cell |
||||
|---|---|---|---|---|---|---|
| cDNA library | A | B | I | II | III | IV |
| Cells for cDNA library construction | 4 million | 4 million | 1 | 1 | 1 | 1 |
| cDNA amplification and barcoding | Oxford Nanopore PCR-cDNA Barcoding kit (SQK-PCB109) | NEBNext® single cell/low input cDNA synthesis & amplification module, NEBNext Ultra II end repair/dA-tailing module, Oxford Nanopore Native Barcoding Expansion (PCR-free) kit (EXP-NBD114), Oxford Nanopore blunt/TA ligase master mix | ||||
| Adapter ligation | Oxford Nanopore Rapid Adapters (RAP; SQK-PCB109) | Oxford Nanopore Adapter Mix II (AMII; EXP-NBD114), NEBNext® quick ligation reaction mix, NEB Quick T4 DNA ligase enzyme | ||||
| Nanopore flow cell | Flongle R9.4.1 | MinION R9.4.1 | ||||
| Sequencer | MinION Mk1C | |||||
| Number of raw reads generated | 1,000,000 | 5,000,000 | ||||
| Total number of pass reads | 305,833 | 195,324 | 678,218 | 1,204,561 | 450,238 | 441,738 |
| Number of antibody transcripts (heavy; light) | 1111; 5122 | 1385; 5463 | 2319; 6281 | 6851; 15133 | 27; 161 | 10; 140 |
| Number of top clone reads (heavy; light) | 461; 2940 | 794; 3795 | 923; 5129 | 3436; 7440 | 5; 114 | 3; 45 |