Build a seqspec (simple)

Overview¶

We will demonstrate the utility of seqspec by examining the structure of reads generated with the 10xv3 single-cell RNA-seq assay on human PBMCs with short read Illumina sequencing.

https://www.10xgenomics.com/datasets/10k-human-pbmcs-3-ht-v3-1-chromium-x-3-1-high

install seqspec download the example spec download the minified reads

`seqspec` structure¶

Assay metadata¶

Library kit: Dual index (possibly nextera) Library protocol: Chromium Single Cell 3’ Reagent Kits User Guide (v3.1 Chemistry Dual Index) (CG000315 Rev C) using the Chromium X Sequence kit:

"NovaSeq 6000 SP Reagent Kit v1.5",
"NovaSeq 6000 S1 Reagent Kit v1.5",
"NovaSeq 6000 S2 Reagent Kit v1.5",
"NovaSeq 6000 S4 Reagent Kit v1.5",

Sequence protocol: Illumina NovaSeq 6000

Library structure¶

Sequence kit primer
Sample index
Sequence primer
Assay structure
- Barcode
- UMI
- polyA
- cDNA
Sequence primer
Sample index
Sequence kit primer

Read structure¶

Paired-end, dual indexing Read 1: 28 cycles (16 bp barcode, 12 bp UMI) i5 index: 10 cycles (sample index) i7 index: 10 cycles (sample index) Read 2: 90 cycles (transcript).

Read 1 contains the 16bp barcode and 12bp UMI i5 contains the 10bp sample index i7 contains the 10bp sample index Read 2 contains 90bp of the cNDA

FASTQ Files