sequencing-base-masks

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