Agent-Skills.md

Agent Skills: Sequencing Read Requirements & 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.

UncategorizedID: CodingKaiser/claude-kaiser-skills/sequencing-base-masks

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CodingKaiser/claude-kaiser-skills
CodingKaiser
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sequencing-base-masks/SKILL.md

Skill Metadata

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 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 |