Why does the SeekArc Tools RNA module not use R1 for alignment in multiome analysis? Does this reduce information content?

Answer:
No. This design is an intentional optimization strategy based on the characteristics of multiome libraries and does not compromise gene expression quantification accuracy.

In multiome experiments, ATAC and RNA libraries are recommended to be sequenced together. Because ATAC libraries contain substantial fixed-sequence content in R1, overall base diversity in R1 is relatively low. Sequencing ATAC libraries alone under these conditions may reduce cluster recognition performance, while mixed-library sequencing can modestly reduce RNA library R1 base quality.

To minimize alignment artifacts introduced by lower-quality R1 bases, the SeekArc Tools RNA module uses: R1 only for cell barcode (CB) and UMI extraction; R2 for transcriptome alignment and quantification. 

The workflow is:

1. Extract CB (17 bp) and UMI (12 bp) from R1 
2. Align R2 reads to the reference genome (STAR recommended) 
3. Perform gene quantification using CB and UMI information 

This strategy improves robustness for standard gene expression analysis and avoids introducing unnecessary alignment noise from lower-quality R1 sequences. Importantly, skipping R1 alignment does not reduce standard gene expression quantification performance.