scg_lib_structs

Single Cell Genomics 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 view the methods. Notes:

  1. The default alignment font is Monaco. Courier New font will be used if Monaco is not available.
  2. Index1 (i7) is always sequenced using the bottom strand as template, regardless of the Illumina machine in use. That is why the index sequences are reverse complementary to the primer sequences.
  3. In a 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 Where RT happens 2nd strand synthesis Full-length cDNA synthesis Barcode addition Pooling before library Library amplification Gene coverage
10x Chromium Single Cell 3’ Droplet In droplets TSO Yes Barcoded RT primers Yes PCR 3’
10x Chromium Single Cell 5’ Droplet In droplets TSO Yes Barcoded TSO primers Yes PCR 5’
BD Rhapsody Nanowells In collection tubes Random priming and primer extension No Barcoded RT primers Yes PCR 3’
CEL-seq/CEL-seq2 FACS In 96w/384w wells RNase H and DNA pol I No Barcoded RT primers Yes In vitro transcription 3’
Drop-seq Droplet In collection tubes TSO Yes Barcoded RT primers Yes PCR 3’
Illumina Bio-Rad SureCell 3’ WTA Droplet In droplets RNase H and DNA pol I No Barcoded RT primers Yes PCR 3’
inDrop Droplet In droplets RNase H and DNA pol I No Barcoded RT primers Yes In vitro transcription 3’
MARS-seq/MARS-seq2.0 FACS In 96w/384w wells RNase H and DNA pol I No Barcoded RT primers Yes In vitro transcription 3’
Microwell-seq Nanowells In collection tubes TSO Yes Barcoded RT primers Yes PCR 3’
Quartz-seq FACS In 96w/384w wells PolyA tailing and primer ligation Yes Ligation of barcoded Truseq adapters No PCR 3’
Quartz-seq2 FACS In 96w/384w wells PolyA tailing and primer ligation Yes Barcoded RT primers Yes PCR 3’
sci-RNA-seq Not needed In situ RNase H and DNA pol I No Barcoded RT primers and library PCR with barcoded primers Yes PCR 3’
sci-RNA-seq3 Not needed In situ RNase H and DNA pol I No Barcoded RT primers and hairpin adapters Yes PCR 3’
scifi-RNA-seq Droplet multiple cells In situ TSO Yes Barcoded RT primers and gel bead barcodes Yes PCR 3’
SCRB-seq/mcSCRB-seq FACS In 96w/384w wells TSO Yes Barcoded RT primers Yes PCR 3’
Seq-Well Nanowells In collection tubes TSO Yes Barcoded RT primers Yes PCR 3’
SMART-seq/SMART-seq2/SMART-seq3 FACS or Fluidigm C1 In 96w/384w wells TSO Yes Library PCR with barcoded primers No PCR full-length
SPLiT-seq Not needed In situ TSO Yes Ligation of barcoded RT primers Yes PCR 3’
STRT-seq FACS In 96w/384w wells TSO Yes Barcoded TSO primers Yes PCR 5’
STRT-seq-C1 Fluidigm C1 In microfluidic chambers TSO Yes Barcoded Tn5 transposase No PCR 5’
STRT-seq-2i FACS or dilution In 9600w wells TSO Yes Barcoded PCR primers and Tn5 transposase Yes PCR 5’

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.


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