Why does SeekOne DD Multiome ATAC + RNA recommend a minimum RNA sequencing configuration of R1 ≥29 bp and R2 ≥90 bp, whereas scFAST-seq workflows recommend PE150?

Answer:
This difference is primarily determined by library structure and downstream analysis requirements.

For scFAST-seq workflows:
The gel beads use semi-random primers consisting of a UMI region plus a 7 bp fixed sequence. After cDNA amplification, the R1 read contains the cell barcode (CB), UMI, and part of the transcript insert.

Longer sequencing reads improve transcript coverage, alignment efficiency, and sensitivity for applications such as: mutation detection, alternative splicing analysis, lncRNA detection, isoform-related analysis. 

Therefore, PE150 is recommended for optimal performance.

For Multiome ATAC + RNA workflows:
The same gel beads are shared between RNA and ATAC libraries. Although the RNA library structure is similar to scFAST-seq, the ATAC library contains substantial fixed-sequence content in R1 derived from transposase adaptor structures, resulting in relatively low base diversity.

Because of this, ATAC libraries should not be sequenced alone and are instead pooled together with RNA libraries. After mixed sequencing, RNA library R1 quality may be modestly affected.

In the SeekArc Tools RNA analysis module:

• R1 is used only for extraction of CB and UMI information (29 bp total) 
• R2 is used for genome alignment and transcript quantification 

As a result:

• R1 only needs sufficient accuracy for barcode and UMI identification 
• R2 requires adequate length for reliable genome alignment 

Therefore, the recommended minimum configuration is: R1 ≥29 bp; R2 ≥90 bp 

Users may still choose PE150 sequencing if preferred. SeekArc Tools natively supports PE150 reads without requiring protocol-specific trimming.