Three Technology Leaders in Single Cell Sequencing: 10X Genomics, Illumina and MissionBio

Reporter: Aviva Lev-Ari, PhD, RN

We review below only Three Technology Leaders in Single Cell Sequencing. There are other players in this research technology space.

WHAT IS SINGLE CELL GENOMICS?

By Nicole Davis, Ph.D.

4.2.4   Three Technology Leaders in Single Cell Sequencing: 10X Genomics, Illumina and MissionBio, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 4: Single Cell Genomics

https://www.jax.org/news-and-insights/2015/december/single-cell-genomics

Platform Solution

Tapestri: The Precision Genomics Platform.

“Single-cell DNA analysis

allows us to actually observe

the clonal composition of

cancer, instead of guessing at

it. This opens up the possibility

of being able to make dynamic

changes in treatment.”

– Catherine Smith, MD, UCSF

Move Beyond Bulk NGS to Precision Genomics

The average read-out from conventional bulk sequencing misses the rare events and underlying genetic

diversity within and across cell populations. To resolve heterogeneity and improve patient stratification,

therapy selection, and disease monitoring we need insights into mutation co-occurrence cell population,

and zygosity within every single cell.

Clonal Resolution with Single-Cell Precision

Complex disease evolves, so understanding genetic variability — including mutation co-occurrence at the single-cell level — is vitally important for clinical researchers to break the cycle of treatment response, resistance and relapse.

CLONAL DIVERSITY REVEALED

High Sensitivity to Reveal True Heterogeneity

The Tapestri Platform revolutionizes the capability to directly assess the clonal architecture of a sample with detection of mutation co-occurrence patterns. Rather than inferring variants that co-occur within a subclone from comparable bulk variant allele frequencies, single-cell resolution uncovers the true distribution of genotypes and their segregation patterns across subclones.

Platform Features

• Targeted and accurate SNVs and indel variant calling
• Single-cell DNA throughput up to 10,000 cells
• Simple workflow
• User-friendly bioinformatics software
• Customizable content
• Detect rare subclones down to 0.1%
• Resolve clonal architecture
• Identify mutation co-occurrence

• How the Tapestri Platform Works

  

  

  

  
  Step 3: After running your sequencer, proceed with our downstream analysis and visualization software. Mission Bio provides dedicated bioinformatics support to help you discover biological and clinical insights

  SOURCE

  https://missionbio.com/tapestri/

  

A highly sensitive method for measuring gene expression from single cells

Generating RNA-Seq libraries from single cell and ultra-low-input samples

Illumina’s Single cell sequencing technologies

Single-Cell Research An Overview of Recent Single-Cell Research Publications Featuring Illumina® Technology

• Quartz-Seq
• Smart-Seq
• Smart-Seq2
• Single-Cell Methylome & Transcriptome Sequencing
• Genome & Transcriptome Sequencing
• Genomic DNA and mRNA Sequencing
• T Cell–Receptor Chain Pairing
• Unique Molecular Identifiers
• Cell Expression by Linear Amplification Sequencing
• Flow Cell–Surface Reverse-Transcription Sequencing
• Single-Cell Tagged ReverseTranscription Sequencing
• Fixed and Recovered Intact Single-Cell RNA Sequencing
• Cell Labeling via Photobleaching Indexing Droplets
• Drop-Seq
• CytoSeq
• Single-Cell RNA Barcoding and Sequencing
• High-Throughput Single-Cell Labeling

TABLE OF CONTENTS

5 Introduction

7 Applications Cancer Metagenomics Stem Cells Developmental Biology Immunology Neurobiology Drug Discovery Reproductive Health Microbial Ecology and Evolution Plant Biology Forensics Allele-Specific Gene Expression 50 Sample Preparation

54 Data Analysis

60 DNA Methods Multiple-Strand Displacement Amplification Genome & Transcriptome Sequencing Multiple Annealing and Looping–Based Amplification Cycles Genomic DNA and mRNA Sequencing

68 Epigenomics Methods Single-Cell Assay for TransposaseAccessible Chromatin Using Sequencing Single-Cell Bisulfite Sequencing/ Single-Cell Whole-Genome Bisulfite Sequencing Single-Cell Methylome & Transcriptome Sequencing Single-Cell Reduced-Representation Bisulfite Sequencing Single-Cell Chromatin Immunoprecipitation Sequencing Chromatin Conformation Capture Sequencing Droplet-Based Chromatin Immunoprecipitation Sequencing

78 RNA Methods Designed Primer–Based RNA Sequencing Single-Cell Universal Poly(A)- Independent RNA Sequencing

PAGES 54 – 56 Data Analysis – most important pages in the Report, $669

Single-cell sequencing poses unique challenges for data analysis. Individual mammalian cells contain 50,000–300,000 transcripts, and gene expression values among individual cells can vary significantly.206 Although several hundred thousand transcripts may be expressed per individual cell, up to 85% of these are present at only 1–100 copies.207 Therefore, it is critically important in scRNA-Seq to capture low-abundance mRNA transcripts and amplify the synthesized cDNA to ensure that all transcripts are ultimately represented uniformly in the library.208,209 Spike-in quantification standards of known abundance can help distinguish technical variability/ noise from biologically meaningful gene expression changes.210 Molecular indexing can also correct for sequencing biases,211.212 and recent improvements in automated sample handling can reduce technical variability even more.213

PAGE 64

Genome & Transcriptome Sequencing

Genome & transcriptome sequencing (G&T-Seq) is a protocol that can separate and sequence genomic DNA and full-length mRNA from single cells.276 In this method, single cells are isolated and lysed. RNA is captured using biotinylated oligo(dT) capture primers and separated from DNA using streptavidin-coated magnetic beads. SmartSeq2 is used to amplify captured RNA on the bead, while MDA is used to amplify DNA. After sequencing, integrating DNA and RNA sequences provides insights into the gene-expression profile of single cells (Table 4)

PAGE 66

Genomic DNA and mRNA Sequencing

DR-Seq studies the genomic and transcriptomic relationship of single cells via sequencing. Nucleic acid amplification prior to physical separation reduces sample loss and the risk of contamination. DR-Seq involves multiple amplification steps, including the quasilinear amplification technique similar to MALBAC. First, mRNAs are reverse-transcribed from lysed single cells using poly(dT) primers with Ad-1x adapters, producing single-stranded cDNA (sscDNA). The Ad-1x adapter sequence contains cell-identifying barcodes, 5’ Illumina adapters, and a T7 promoter. Next, both gDNA and sscDNA are amplified simultaneously via quasilinear WGA with Ad-2 primers. These primers are similar to MALBAC adapters, containing 8 random nucleotides for random priming followed by a constant 27-nucleotide

PAGE 73

Single-Cell Methylome & Transcriptome Sequencing

scM&T-Seq allows parallel analysis of both epigenetic and gene expression patterns from single cells using Smart-Seq2 and scBS-Seq. scM&T-Seq is built upon G&T-Seq, but instead of using MDA for DNA sequencing, it uses scBS-Seq to interrogate DNA methylation patterns. First, single cells are isolated and individually lysed. Then, mRNAs are isolated using streptavidin-coupled mRNA capture primers, physically separating them from DNA strands. Smart-Seq2 is used to generate cDNA libraries from the mRNA, which involves reverse transcription with template switching and tagmentation. DNA libraries are prepared via scBS-Seq, which involves bisulfite conversion of DNA strands to identify methylated cytosines. Both libraries are now ready for sequencing (Table 9).

PAGE 78

RNA METHODS

Low-level RNA detection refers to both detection of rare RNA molecules in a cellfree environment (such as circulating tumor RNA) and the expression patterns of single cells. Tissues consist of a multitude of different cell types, each with a distinctly different set of functions. Even within a single cell type, the transcriptomes are highly dynamic and reflect temporal, spatial, and cell cycle–dependent changes. Cell harvesting, handling, and technical issues with sensitivity and bias during amplification add additional levels of complexity. To resolve this multitiered complexity would require analyzing many thousands of cells. The use of unique barcodes has greatly increased the number of samples that can be multiplexed and pooled at little to no decrease in reads associated with each sample. Recent improvements in cell capture and sample preparation will provide more information, faster, and at lower cost.310,311 This development promises to expand our understanding of cell function fundamentally, with significant implications for research and human health. 312

PAGR 87

Genome & Transcriptome Sequencing

G&T-Seq is a protocol that can separate and sequence genomic DNA and full-length mRNA from single cells.327 In this method, single cells are isolated and lysed. RNA is captured using biotinylated oligo(dT) capture primers and separated from DNA using streptavidin-coated magnetic beads. Smart-Seq2 is used to amplify captured RNA on the bead, while MDA is used to amplify DNA. After sequencing, integrating DNA and RNA sequences provides insights into the gene-expression profile of single cells.

PAGE 88

Genomic DNA and mRNA Sequencing

DR-Seq studies the genomic and transcriptomic relationship of single cells via sequencing. Nucleic acid amplification prior to physical separation reduces sample loss and the risk of contamination. DR-Seq involves multiple amplification steps, including the quasilinear amplification technique similar to MALBAC.

PAGE 89

T Cell–Receptor Chain Pairing

Functional TCRs are heterodimeric proteins composed of unique combinations of α and β chains. For an accurate functional analysis, both subunits must be sequenced together to avoid disrupting the α- and β-chain pairing during the cell lysis step.333

PAGE 91

Flow Cell–Surface Reverse-Transcription Sequencing

Flow cell–surface reverse-transcription sequencing (FRT-Seq) is a transcriptomesequencing technique developed in 2010.339 It is strand-specific, free of amplification, and is compatible with paired-end sequencing. To begin with, poly(A)+ RNA samples are fragmented by metal-ion hydrolysis and dephosphorylated.

PAGE 97

Single-Cell RNA Barcoding and Sequencing

Single-cell RNA barcoding and sequencing (SCRB-Seq) is a cost-efficient, multiplexed scRNA-Seq technique. SCRB-Seq isolates single cells into wells using FACS. After cell lysis, poly(A)+ mRNAs are annealed to a custom primer containing a poly(T) tract, UMI, well barcode, and biotin. Template-switching reverse transcription and PCR amplification are carried out on the mRNA, generating barcoded full-length cDNA. cDNA strands from all wells are pooled together to be purified. They are amplified by PCR and purified further. cDNA libraries are prepared using the Nextera XT kit with modified i5 primers. The resultant cDNA fragments are size-selected for 300–800 bp and sequenced (Table 31).

https://www.illumina.com/content/dam/illumina-marketing/documents/products/research_reviews/single-cell-sequencing-research-review.pdf

Scientific Publication Reviews can be accessed at

http://www.illumina.com/pubreviews

SOURCE

https://www.illumina.com/techniques/sequencing/rna-sequencing/ultra-low-input-single-cell-rna-seq.html

https://www.10xgenomics.com/company/#about

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accelerate science

10x Genomics is building tools for scientific discovery that reveal and address the true complexities of biology and disease. Through a combination of novel microfluidics, chemistry and bioinformatics, our award-winning Chromium System is enabling researchers around the world to more fully understand the fundamentals of biology at unprecedented resolution and scale.

Single Cell Technology

Revolutionizing Gene Expression

WITH SINGLE-CELL RNA-SEQ

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