scg_lib_structs

Single Cell RNA-seq (scRNA-seq) Library Structure

Collections of library structure and sequence of popular single cell genomic methods (mainly scRNA-seq).

How to use?

Click the following links to veiw the methods. Notes:

  1. the default alignment font is Monaco. Courier New font will be used if Monaco is not available.
  2. In a pair-end and dual-index library, how index2 (i5) is sequenced differs from machines to machines. According to the Index Sequencing Guide from Illumina, Miseq, Hiseq2000/2500 and NovaSeq 6000 use the bottom strand as template, which is why the index sequences are the same as the primer sequences in those machines. iSeq 100, MiniSeq, NextSeq, HiSeq X and HiSeq 3000/4000 use the top strand as template, which is why the index sequences are reverse-complementary to the primer sequences in those machines. All methods listed below use Miseq, Hiseq2000/2500 and NovaSeq as examples.

Technical comparisons (scRNA-seq only)

The basic chemistry is very similar, the main differences among those scRNA-seq methods are summarised in the table below. For a detailed discussion, check the text boxes from our review: From Tissues to Cell Types and Back: Single-Cell Gene Expression Analysis of Tissue Architecture

  Single cell isolation/capture 2nd strand synthesis Full-length cDNA synthesis Barcode addition Pooling before library Library amplification Gene coverage
10x Chromium Single Cell 3’ Droplet TSO Yes Barcoded RT primers Yes PCR 3’
10x Chromium Single Cell 5’ Droplet TSO Yes Barcoded TSO primers Yes PCR 5’
BD Rhapsody Nanowells Random priming and primer extension No Barcoded RT primers Yes PCR 3’
CEL-seq/CEL-seq2 FACS RNase H and DNA pol I No Barcoded RT primers Yes In vitro transcription 3’
Drop-seq Droplet TSO Yes Barcoded RT primers Yes PCR 3’
MARS-seq FACS RNase H and DNA pol I No Barcoded RT primers Yes In vitro transcription 3’
Microwell-seq Nanowells TSO Yes Barcoded RT primers Yes PCR 3’
Quartz-seq FACS PolyA tailing and primer ligation Yes Ligation of barcoded Truseq adapters No PCR 3’
Quartz-seq2 FACS PolyA tailing and primer ligation Yes Barcoded RT primers Yes PCR 3’
SMART-seq/
SMART-seq2
FACS or Fluidigm C1 TSO Yes Library PCR with barcoded primers No PCR full-length
SPLiT-seq Not needed TSO Yes Ligation of barcoded RT primers Yes PCR 3’
STRT-seq FACS TSO Yes Barcoded TSO primers Yes PCR 5’
STRT-seq-C1 Fluidigm C1 TSO Yes Barcoded Tn5 transposase No PCR 5’
STRT-seq-2i FACS or dilution TSO Yes Barcoded PCR primers and Tn5 transposase Yes PCR 5’
Seq-Well Nanowells TSO Yes Barcoded RT primers Yes PCR 3’
Illumina Bio-Rad SureCell 3’ WTA Droplet RNase H and DNA pol I No Barcoded RT primers Yes PCR 3’
inDrop Droplet RNase H and DNA pol I No Barcoded RT primers Yes In vitro transcription 3’
SCRB-seq/
mcSCRB-seq
FACS TSO Yes Barcoded RT primers Yes PCR 3’
sci-RNA-seq Not needed RNase H and DNA pol I No Barcoded RT primers and library PCR with barcoded primers Yes PCR 3’

Motivation

I was a little bit bombarded with all the single cell methods and got completely lost. To help myself understand all of them and future troubleshooting, I start to perform an on-paper library preparation whenever I see a new single cell method.

Why bother?

Here I borrow from Feyman:

What I cannot create, I do not understand.


TODO:

Feedback

I would be very happy if you go through them and let me know what you think. If you spot some errors/mistakes, or I’ve missed some key methods. Feel free to contact me:

Xi Chen
chenx9@sustech.edu.cn