10x Chromium Single Cell 3' Gene Expression v2

Droplet single-cell RNA-seq — the generation before v3. Every droplet holds one gel bead, which tags all of a cell's transcripts with the same cell barcode and each individual mRNA molecule with a unique UMI. That lets you count molecules per gene per cell.

How it's read

Two reads come off the sequencer:

read	length	what it holds
R1	26 bp	a 16 bp cell barcode + a 10 bp UMI
R2	open-ended	the cDNA (the transcript itself), read sense to the mRNA

The cell barcode is drawn from 10x's published 737K-august-2016 list (~737,000 barcodes).

How seqforge tells v2 apart from v3

The only difference visible in the reads is R1 length: v2 is 16 + 10 = 26 bp; v3 is 16 + 12 = 28 bp. That 2 bp gap is decisive on its own, so seqforge separates v2 from v3 by read geometry alone — before it ever consults a barcode list. The two are never confused. (Their whitelists differ too — 737K-august-2016 vs 3M-february-2018 — but length settles it first.)

If a paper or database says "v2" while the reads are actually 28 bp, seqforge does not quietly pick one. It surfaces the disagreement (metadata says 26 bp, the bytes say 28 bp) and hands it to a human — the bytes decide what the data is.

The one neighbour the reads cannot decide

5' v1/v2 is this entry's twin in every byte. It has the same 26 bp R1 (16 bp barcode + 10 bp UMI), the same open-ended cDNA R2, and — the part that closes every cheap route — the same 737K-august-2016 whitelist. Both entries point at the same file, so the barcode list cannot break the tie either. The backends differ in exactly one parameter, soloStrand, and no probe can observe it: 5' reads the transcript from the other end.

That makes it the only read-undecidable pair in this knowledge base, and worth knowing because the resolver's behaviour changes here. Rather than pick, it reaches for metadata (papers say "5'" or "3'" reliably even when they are vague about the version) or a trial alignment, and asks a human if neither answers. Before the 5' entry existed a 5' library resolved silently to this spec and compiled soloStrand Forward — a measured 0.5–0.6 gene-expression correlation against >0.98 for the right orientation, at exit 0, with nothing red.

None of this is true of v3: at 28 bp its geometry-mates each carry their own list, so the barcode list settles them.

Gotchas

• The barcode read can go missing on SRA. fasterq-dump without --include-technical drops R1 entirely; seqforge blocks rather than guessing which file is which. Re-fetch with --include-technical, or pull the submitter's original files.
• SRA rewrites read-name headers, so seqforge never trusts them when grouping files into samples.

References

Read structure cross-checked against scg_lib_structs — 10x Chromium 3' (Teichmann Lab, CC-BY), which lays out the v2–v4 kits side by side. The exact, machine-readable definition seqforge uses lives in this entry's spec.yaml.