sequencing-base-masks
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.
What this skill does
# Sequencing Read Requirements & Base Masks ## Key concept: `I` vs `y` - **`I` (capital)** = output as index FASTQ (I1/I2) — used for sample demultiplexing - **`y` (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 |
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