📚 Adapter Schemes
CutSeq supports various built-in adapter schemes for different NGS library preparation methods. Each scheme follows a general pattern:
Components
- p5: 5’ Illumina sequencing adapter (shown in light green)
- p7: 3’ Illumina sequencing adapter (shown in pale green)
- inline5: Fixed DNA barcode sequences in brackets () (shown in light cyan)
- inline3: Fixed DNA barcode sequences in brackets () (shown in light sky blue)
- umi5: 5’ Random UMI sequences marked as N (shown in dodger blue)
- umi3: 3’ Random UMI sequences marked as N (shown in steel blue)
- mask5: Sequences to be masked marked as X (shown in gainsboro)
- mask3: Sequences to be masked marked as X (shown in light gray)
- strand: Direction indicator (shown with >, <, or -)
Built-in Schemes
DSLIGATION (dsDNA Ligation)
AGTTCTACAGTCCGACGATC
>
AGATCGGAAGAGCACACGTC - Basic dsDNA ligation with A-tailing
- Forward orientation
- No UMIs or special trimming needed
SMALLRNA (Small RNA Libraries)
AGTTCTACAGTCCGACGATC
>
AGATCGGAAGAGCACACGTC - Used for small RNA sequencing
- Double ligation method
- Forward orientation
- Optional 2nt trimming on both ends for quality
INLINE (Custom Barcoded Libraries)
AGTTCTACAGTCCGACGATC NNNNN
>
NNNNN (ATCACG) AGATCGGAAGAGCACACGTC - Used for libraries with inline barcodes
- Dual UMI design (5nt each)
- Forward orientation
- Contains fixed barcode sequence
TAKARAV2 (SMARTer® Stranded Protocol V2)
ACACGACGCTCTTCCGATCT XXXXXX
<
XXXXXX AGATCGGAAGAGCACACGTC - Handles polyC/G artifacts from random RT priming
- Extended masking for adaptase tail (up to 15bp)
- Reverse orientation
- Uses random polyC tail as pseudo-UMI
PBAT (Post-Bisulfite Adapter Tagging)
ACACGACGCTCTTCCGATCT XXXXXX
<
XXXXXX AGATCGGAAGAGCACACGTC - Used for post-bisulfite DNA sequencing
- Random primer-based adapter addition
- Reverse orientation
- Symmetric masking for random tails
NEXTERA (ATAC-seq)
AGATGTGTATAAGAGACAG
>
CTGTCTCTTATACACATCT - Used for ATAC-seq libraries
- Simple design without UMIs or barcodes
- Forward orientation
- Standard Nextera adapters
ILLUMINARNA (Illumina Stranded RNA-Seq)
AGATGTGTATAAGAGACAG
<
CTGTCTCTTATACACATCT - Standard Illumina stranded RNA-seq protocol
- Reverse orientation
- Simple design without UMIs or masking
- Direct adapter ligation method
STRANDED (Stranded RNA Library)
ACACGACGCTCTTCCGATCT X
<
XXX AGATCGGAAGAGCACACGTC - Reverse orientation
- For SMARTer-type stranded RNA-seq
- Minimal masking on 5’ and 3’ ends
TAKARAV3 (SMARTer® Pico v3)
ACACGACGCTCTTCCGATCT XXX
<
XXXXXX NNNNNNNN AGATCGGAAGAGCACACGTC - Reverse orientation
- SMARTer v3 protocol with complex UMI linker
- 14-nt UMI used for molecule tracking
ECLIP6 (eCLIP or SAC-seq with 6nt UMI)
ACACGACGCTCTTCCGATCT XX
<
X NNNNNN AGATCGGAAGAGCACACGTC - Reverse orientation
- 6-nt UMI for identifying PCR duplicates
- Common in SAC-seq or eCLIP protocols
ECLIP10 (eCLIP with 10nt UMI)
ACACGACGCTCTTCCGATCT XX
<
X NNNNNNNNNN AGATCGGAAGAGCACACGTC - Reverse orientation
- 10-nt UMI improves molecule resolution
- Used in eCLIP or high-depth cDNA methods
SACSEQV3 (SAC-seq with dual UMIs)
ACACGACGCTCTTCCGATCT NNNNNNNN X
>
XX NNNNNNNN AGATCGGAAGAGCACACGTC - Dual-UMI design (8 nt each side)
- cDNA ligation-based protocols
- Used in SAC-seq v3 and similar workflows (designed by YC)
XGENRNA (xGen RNA with polyG/C tails)
ACACGACGCTCTTCCGATCT XXXXXX
<
XXXXXXXXXXXXXXX AGATCGGAAGAGCACACGTC - Reverse orientation
- Random RT primer artifacts masked
- Adaptase tail trimmed (up to 15bp)
XGENMETHY (xGen Methyl-seq)
ACACGACGCTCTTCCGATCT XX
>
XXXXXXXXXX AGATCGGAAGAGCACACGTC - Forward orientation
- Trim 2nt from R1 start and 10nt from R2 start
- Designed for bisulfite-converted methylation libraries
XGENSNMC (xGen snmC-seq)
ACACGACGCTCTTCCGATCT XXXXXX
>
XXXXXXXXXXXXXXX AGATCGGAAGAGCACACGTC - Forward orientation
- For single-cell bisulfite sequencing
- 15bp trimming due to RT or ligation artifact