10x Chromium Single Cell 3' Gene Expression v3 (and v3.1)

Droplet single-cell RNA-seq — the current-generation 3' kit. Every droplet holds one gel bead, which tags all of a cell's transcripts with the same cell barcode and each mRNA molecule with a unique UMI.

This page covers both v3 and v3.1. To a sequencer they are the same library, and seqforge processes them identically — see v3 vs v3.1 below.

How it's read

read	length	what it holds
R1	28 bp	a 16 bp cell barcode + a 12 bp UMI
R2	open-ended	the cDNA (the transcript itself), read sense to the mRNA

The cell barcode is drawn from 10x's 3M-february-2018 list (~6.8 million barcodes). Index reads (I1/I2) may or may not be present; seqforge doesn't need them.

Names you'll see in the wild: "10x 3' v3", "Chromium 3' v3", "SC3Pv3", "single cell 3-prime v3", "Next GEM 3' v3.1". Papers often write only "10x 3' v3" for a v3.1 run, or name the kit and not the version — which changes nothing, as the next section explains.

v3 vs v3.1

They are identical to seqforge: same read layout, same barcode list, same alignment settings. No probe can tell them apart from the reads, and none needs to — so seqforge records both names and asks you nothing. v3.1 exists as its own knowledge-base entry only so that "these two really are identical" is something the test suite re-checks on every build, rather than a claim sitting in a comment.

How seqforge tells it apart from other chemistries

• v2 — a 16 bp barcode + a 10 bp UMI makes a 26 bp R1 (vs v3's 28 bp), on a different list. The 2 bp length difference alone separates them.
• Multiome (ARC) and GEM-X 3' v4 produce the same 28 bp / 16+12 layout — here only the barcode list tells them apart, because each uses a different one. Geometry narrows to a family; the list picks the exact member.
• 5' kits share the barcode/UMI geometry but read the cDNA in the opposite direction. The reads can't reveal that, so it takes metadata or a trial alignment, not geometry.

Gotchas

• The barcode read can go missing on SRA. fasterq-dump without --include-technical drops the 28 bp R1; seqforge blocks rather than inventing a barcode read from a filename. Re-fetch with --include-technical, or pull the submitter's original files.
• A pre-trimmed upload (R1 no longer a single fixed length) shifts the barcode offsets — seqforge refuses rather than reading the barcode from the wrong place.
• 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). The exact, machine-readable definition seqforge uses lives in each entry's spec.yaml.