BD Rhapsody WTA (Whole Transcriptome Analysis)

BD Biosciences' droplet-free single-cell 3' RNA-seq. Cells are captured in a microwell plate and tagged with a cell label built from three barcode blocks on the capture bead, plus a UMI on each molecule.

BD ships this chemistry in two bead generations, and seqforge recognizes and compiles both automatically, from the reads. The original fixed-position bead is described first; the 2022 Enhanced bead — with its variable-length diversity insert — is described below. seqforge tells the two apart from the bytes (their linker structure differs), so you never have to declare which bead a dataset used.

How it's read

Two reads:

• R2 = the cDNA (the transcript), read sense to the mRNA.
• R1 = the bead read: three 9 bp cell-label blocks separated by two fixed linker sequences, then an 8 bp UMI.

segment on R1	length	position
cell label 1	9 bp	0–9
linker 1 (ACTGGCCTGCGA)	12 bp	9–21
cell label 2	9 bp	21–30
linker 2 (GGTAGCGGTGACA)	13 bp	30–43
cell label 3	9 bp	43–52
UMI	8 bp	52–60

Each cell-label block is drawn from its own published list of 97 9-mers, so there are 97³ ≈ 913,000 possible cell labels. R1 is often sequenced past position 60 into poly(T)/cDNA; those extra bases are ignored.

How seqforge tells it apart

The two fixed linker sequences at known positions are the signature — no other supported chemistry has them. Each cell-label block also matches its own barcode list, which both confirms the call and separates BD Rhapsody from a plain bulk paired-end library (bulk has no barcode list to match).

Strand

The bead's capture oligo ends in poly(dT), which primes off the mRNA poly(A) tail, so R2 reads the cDNA sense to the transcript — a standard 3' poly-dT library, like 10x 3'. Getting this backwards would leave most reads uncounted while the aligner still exits cleanly, so seqforge derives it from the bead chemistry rather than guessing.

Enhanced beads (2022)

In 2022 BD introduced Enhanced Cell Capture Beads, the current chemistry. The cell labels work the same way — three 9 bp blocks read off the bead — but two things change:

• A short diversity insert of 0–3 bp (one of nothing, A, GT, or TCA) is added at the very start of Read 1, to stagger the reads and cut the amount of PhiX needed.
• The two linkers shrink to GTGA and GACA (4 bp each), from v1's 12 and 13 bp.

So Enhanced Read 1 reads:

[0–3 bp insert] [CLS1 · 9] GTGA [CLS2 · 9] GACA [CLS3 · 9] [UMI · 8]

There are two sub-versions, differing only in the cell-label lists: 96 or 384 sequences per block (the 384-list "Enhanced v2" allows more cell labels). The read layout is otherwise identical.

How seqforge handles the Enhanced bead

seqforge recognizes and compiles the Enhanced bead automatically — you do not tell it the bead generation, and you do not pre-trim the diversity insert.

• Recognition. The diversity insert shifts every barcode by a different amount from one read to the next, so the positions are not fixed. seqforge finds the GTGA…GACA linker frame in each read (tolerating the 0–3 bp stagger and sequencing error), then reads the cell-label and UMI blocks relative to it. The presence of that frame is what separates Enhanced from the original bead, whose longer ACTGGCCTGCGA/GGTAGCGGTGACA linkers sit at fixed positions instead.
• 96 vs 384. The two Enhanced sub-versions have identical read layout and differ only in which cell-label list their blocks match, so seqforge picks the one the barcodes actually hit — exactly how it tells 10x v2 from v3. The 96-plex bead reuses the original bead's cell-label lists; the 384-plex lists ship with seqforge too.
• Compilation. seqforge maps Enhanced reads with STARsolo's adapter anchor — no pre-trimming — and derives the exact settings STAR's author endorsed in issue #1607 straight from the read layout:

STAR --soloType CB_UMI_Complex \
     --soloAdapterSequence NNNNNNNNNGTGANNNNNNNNNGACA \
     --soloCBposition 2_0_2_8 2_13_2_21 3_1_3_9 \
     --soloUMIposition 3_10_3_17 \
     --soloCBwhitelist BD_CLS1.txt BD_CLS2.txt BD_CLS3.txt

The adapter NNNNNNNNNGTGANNNNNNNNNGACA is CLS1(9) + GTGA + CLS2(9) + GACA; STARsolo finds it in each read and reads the barcodes relative to it, so the variable insert at the front takes care of itself. For a reproducible reference workflow spanning every bead generation, see rhapsodist.

References

Read structure, linker sequences, and barcode lists pinned from scg_lib_structs — BD Rhapsody (Teichmann Lab, CC-BY), cross-checked against the seqspec bd_rhapsody_v1 example and the STARsolo maintainer's endorsed settings. The exact, machine-readable definition seqforge uses lives in this entry's spec.yaml.