| name | sequencing-base-masks |
| description | Sequencing read requirements and bcl2fastq base masks for 10x Genomics libraries: chemistry-specific BasesMaskCR values, ATAC I2 barcode requirement (capital I not lowercase y), Visium FFPE vs frozen R2 length difference. |
Sequencing Read Requirements & Base Masks
Key concept: I vs y
I (capital) = output as index FASTQ (I1/I2) — used for sample demultiplexingy (lowercase) = output as regular read FASTQ (R1, R2, R3…)
For 10x ATAC, the i5 read IS the cell barcode — must use capital I to produce I2 FASTQ, or CellRanger ARC fails.
Base Masks by Chemistry
3' / 5' Gene Expression
| Chemistry | BasesMaskCR |
|---|
| 3' v2 (legacy) | y26n*,I8n*,y* |
| 3' v3/v4/GEM-X | y28n*,I10n*,I10n*,y* |
| 5' v2/HT (legacy) | y26n*,I10n*,I10n*,y* |
| 5' v3 / Universal 5' | y28n*,I10n*,I10n*,y* |
| 5' R1-only | y41n*,I10n*,I10n* |
| 5' R1-only v3 | y43n*,I10n*,I10n* |
VDJ / Feature Barcoding / CellPlex
| Chemistry | BasesMaskCR |
|---|
| VDJ v2/v3 | y28n*,I10n*,I10n*,y91n* |
| Feature Barcode (CITE-seq) | y28n*,I10n*,I10n*,y* |
| CellPlex / CMO | y28n*,I10n*,I10n*,y* |
ATAC & Multiome
| Chemistry | BasesMaskCR | Notes |
|---|
| ATAC solo v1/v2 | y50n*,I8n*,I16n*,y50n* | i5=16bp barcode → capital I |
| Multiome GEX | y28n*,I10n*,I10n*,y* | |
| Multiome ATAC | y50n*,I8n*,I24n*,y49n* | i5=24bp (16bp BC + 8bp linker) → capital I |
Do NOT pool ATAC libraries with other 10x types — incompatible i5.
Spatial (Visium)
| Chemistry | BasesMaskCR | Notes |
|---|
| Visium frozen | y28n*,I10n*,I10n*,y90n* | Kit TT |
| Visium FFPE | y28n*,I10n*,I10n*,y50n* | R2=50bp — shorter insert |
| Visium CytAssist | y28n*,I10n*,I10n*,y90n* | Same as frozen (NOT FFPE) |
| Visium HD | y43n*,I10n*,I10n*,y151n* | Extended R1 |
Fixed RNA (Flex)
| Chemistry | BasesMaskCR |
|---|
| Flex GE v2 standard | y28n*,I10n*,I10n*,y90n* |
| Flex GE v2 R1-long | y54n*,I10n*,I10n*,y50n* |
| Flex Protein Expression | y28n*,I10n*,I10n*,y90n* |
Recommended Sequencing Depths
| Chemistry | Depth |
|---|
| 3' / 5' GEX, Multiome GEX | ≥20,000 pairs/cell |
| ATAC solo / Multiome ATAC | ≥25,000 pairs/nucleus |
| Visium frozen / CytAssist | ≥50,000 pairs/spot |
| Visium FFPE | ≥25,000 pairs/spot |
| Flex GE | ≥5,000–10,000 pairs/cell |
