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SPLiT-seq (split-pool combinatorial barcoding)

A single-cell RNA-seq method that needs no special instrument. Instead of one droplet per cell, cells are barcoded by splitting and pooling them through several rounds of labeling; after three rounds each cell carries a unique combination of three barcodes.

This entry covers the original published SPLiT-seq (Rosenberg et al., Science 2018). Parse Biosciences' Evercode is a separate commercial descendant with different linkers and barcodes — a different chemistry, not covered here.

How it's read

  • Read 1 = the cDNA (the transcript).
  • Read 2 = the barcode read: a 10 bp UMI, then three 8 bp round-barcodes separated by two fixed 30 bp linkers.
[10 bp UMI] [8 bp round-3] --linker1-- [8 bp round-2] --linker2-- [8 bp round-1]

A cell's identity is the combination of its round-1, round-2, and round-3 barcodes; each round draws from a list of ~96 barcodes.

How seqforge tells it apart

Like BD Rhapsody, the two fixed linker sequences at known positions are the signature, and each round-barcode matches its own list. The exact linker lengths matter: the original 2018 chemistry puts round-1 at a different position than the later Parse chemistry, so a position copied from the wrong source silently mismatches. seqforge pins the original layout from the paper's own oligos.

Status: one question still open

SPLiT-seq is the pilot's generalization test — it exercises machinery the 10x entries don't (8 bp barcodes instead of 16, a barcode split combinatorially across a read). All three round-barcode lists ship packed with seqforge, so the entry resolves and compiles from bytes alone. One thing is still unconfirmed:

  • The strand has not been checked against real data. It's derived from the paper's oligos and corroborated by the authors' own pipeline (both point the same way, and no source anywhere claims the opposite), but the honest state is that most pipelines never explicitly chose the strand — they inherited a default, so agreement with them is weaker evidence than it looks. The decisive check is to run the paper's own data both ways; the correct strand assigns roughly an order of magnitude more reads.

References

Read structure and linker sequences pinned from scg_lib_structs — SPLiT-seq (Teichmann Lab, CC-BY) and independently reconstructed from the paper's oligo tables (Rosenberg et al., Science 2018). The exact, machine-readable definition seqforge uses lives in this entry's spec.yaml.