Are the feature barcode oligo conjugation's random nucleotides (Ns) required in the feature barcode oligo conjugation?

Question:  Are the feature barcode oligo conjugation's random nucleotides (Ns) required in the feature barcode oligo conjugation?

Answer:  Our testing in house utilized the oligo structure published in our Demonstrated Protocol: Cell Surface Protein Labeling for Single Cell RNA Sequencing Protocols.

While excluding the random bases will still enable the successful production of cell surface protein libraries, we do still recommend keeping the N's as they:

1. Help to increase the length of the final library construct (making it > 200bp)
2. Add diversity stretches, which is beneficial if the libraries are sequenced without pooling with their corresponding gene expression libraries.
3. Improve sequencing quality by giving the sequencers random bases to calibrate base-calling before mono-template issues with the feature barcode

Note: The final performance with or without Ns was similar when comparing the quality differences in Antibody Application metrics (contained in the web summary output). Also, if the N’s are omitted, the feature reference file will need to be adjusted to take this into account.

Please refer to our support website for information on Feature Barcoding analysis:

Single Cell 3' v3/v3.1:

https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/feature-bc-analysis 

 Single Cell VDJ:

https://support.10xgenomics.com/single-cell-vdj/software/vdj-and-gene-expression/latest/overview

Products: Single Cell Gene Expression, Single Cell Immune Profiling