compare singe cell isolation
compare singe cell isolation
Our solution provides you with a faster, more cost-effective, and efficient instrument for single-cell partitioning compared to others
SeekOne® Digital Droplet System
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Overview of SeekOne® Digital Droplet System
SeekOne® Digital Droplet System (SeekOne® DD System), self-developed by Beijing SeekGene® Biotechnology Co., Ltd., can be applied to various medical studies, including cancer, immunity, cell development, viral infection, drug discovery, and target screening. Based on microfluidic technology, the system achieves single-cell partitioning and capture with water-in-oil droplets. Barcoded beads are designed for the molecular labelling of different cells and their mRNAs. After labelling single cells for gene expression, high- throughput single-cell libraries can be constructed. Finally, SeekSoul® Tools, our efficient single-cell analysis software, can be used to process and analyze single-cell data.
2. Highlights for SeekOne® DD
- SeekOne® Digital Droplet System is an automated instrument for single cell partitioning, capture, and labelling based on the principle of
- The DD system is tailored for highly viable cells, such as brain tissue, retinal tissue, and all tissues from aged individuals.
- The DD system is compatible with various single cell products that facilitate a range of single cell experiments, including single nuclei RNA- sequencing, single-cell 3’ transcriptome-seq, single-cell 5’ transcriptome-seq, single-cell full-
length RNA sequence transcriptome-seq, and single cell immune profiling with many more on the way.
- High sample throughput of up to 8 samples in parallel is suitable for large cohort or atlas studies where multiple samples need to be processed simultaneously.
- Equipped with a temperature control system that ensures more stable reactions, especially for laboratories with a high temperature variance from room temperature.
Product Features
Fast: Rapidly generates 150,000 water-in-oil droplets in 3 minutes, reducing cell loss.
Efficient: Up to 12,000 cells can be captured in a single channel with high cell capture rates of up to 65%. Flexible running of 1~8 samples in parallel. No need to manually add glycerol to empty wells.
Accurate: Low doublet rates of less than 0.3% per 1,000 cells; equipped with a temperature control system to eliminate the effects of environmental temperature differences.
Compatible: Highly compatible system with a real- time remote upgrade can be used with comprehensive SeekOne® product lines to meet a variety of scientific research needs.
User-friendly: Small instrument with large screen; 7″
+ 45° view touch interface makes “one-click” operation more convenient and comfortable.
Cost-saving: Vacant channels can be occupied by Place Chip without wasting chips.
Working Principle of SeekOne® DD System
SeekOne® DD system performs single-cell partition, capture, and labelling using microfluidic digital droplets and barcoded beads.
Microfluidic digital droplet: -shaped channel
Figure 1. Core technology of SeekOne® DD System. Cells and barcoded beads are added separately and then react in the carrier oil to form emulsion droplets in the -shaped channel. After that, mRNA molecules released by the cells are captured by oligo(dT) on the barcoded beads.SeekOne® DD system offers a one-stop-shop single-cell solution from single-cell partition, capture, labelling, and library construction to data analysis.
Figure 2. SeekOne® DD System Workflow. The process begins with the collection of single cell suspension. After single cell partitioning, capture and labelling, a single cell library compatible with Illumina and MGI sequencers is constructed for high-throughput sequencing. Data can be processed using SeekSoul® Tools ——the efficient data analysis software to explore cell heterogeneity.
Rapid generation of 150,000 water-in-oil droplets in 3 minutes
- Efficiently capture 500-12,000 cells per channel
- Flexible running of 1~8 samples in parallel
- Cell size flexibility: cell diameter of 5~40 μm
- High cell capture rates of up to 65%
- Low doublet rates of under 3% per 1,000 cells
Instrument
Compatible Kits
SeekOne® DD Single Cell Full-length RNA Sequence Transcriptome-seq (scFAST-seq) Kit
| Product | Product code |
| SeekOne® DD Single Cell Full-length RNA Sequence Transcriptome-seq (scFAST-seq), 2 tests/8 tests | K00801-02/K00801-08 |
| Product Components | Component code |
| SeekOne® DD Chip S3 Kit, 2 tests/8 tests | K00202-0201/K00202-0801 |
| SeekOne® DD scFAST-seq Barcoded Beads Kit, 2 tests/8 tests | K00801-0202/K00801-0802 |
| SeekOne® DD scFAST-seq Reverse Transcription Kit, 2 tests/8 tests | K00801-0203/K00801-0803 |
| SeekOne® DD Library Construction Kit, 2 tests/8 tests | K00202-0204/K00202-0804 |
| SeekOne® DD scFAST-seq Cleanup Kit, 2 tests/8 tests | K00202-0205/K00202-0805 |
SeekOne® DD Single Cell 3’ Transcriptome-seq Ki
| Product | Product code |
| SeekOne® DD Single Cell 3’ Transcriptome-seq Kit, 2 tests/8 tests | K00202-02/K00202-08 |
| Product Components | Component code |
| SeekOne® DD Chip S3 Kit, 2 tests/8 tests | K00202-0201/K00202-0801 |
| SeekOne® DD Single Cell 3’ Barcoded Beads Kit, 2 tests/8 tests | K00202-0202/K00202-0802 |
| SeekOne® DD Single Cell 3’ Reverse Transcription Kit, 2 tests/8 tests | K00202-0203/K00202-0803 |
| SeekOne® DD Library Construction Kit, 2 tests/8 tests | K00202-0204/K00202-0804 |
| SeekOne® DD Single Cell Cleanup Kit, 2 tests/8 tests | K00202-0205/K00202-0805 |
SeekOne® DD Single Cell 3’ Transcriptome-seq Kit
| Product | Product code |
| SeekOne® DD Single Cell 3’ Transcriptome-seq Kit, 2 tests/8 tests | K00202-02/K00202-08 |
| Product Components | Component code |
| SeekOne® DD Chip S3 Kit, 2 tests/8 tests | K00202-0201/K00202-0801 |
| SeekOne® DD Single Cell 3’ Barcoded Beads Kit, 2 tests/8 tests | K00202-0202/K00202-0802 |
| SeekOne® DD Single Cell 3’ Reverse Transcription Kit, 2 tests/8 tests | K00202-0203/K00202-0803 |
| SeekOne® DD Library Construction Kit, 2 tests/8 tests | K00202-0204/K00202-0804 |
| SeekOne® DD Single Cell Cleanup Kit, 2 tests/8 tests | K00202-0205/K00202-0805 |
SeekOne® DD Single Cell 5’ Transcriptome-seq Kit
| Product | Product code |
| SeekOne® DD Single Cell 5’ Transcriptome-seq Kit, 2 tests/8 tests | K00501-02/K00501-08 |
| Product Components | Component code |
| SeekOne® DD Chip S3 Kit, 2 tests/8 tests | K00202-0201/K00202-0801 |
| SeekOne® DD Single Cell 5’ Barcoded Beads Kit, 2 tests/8 tests | K00501-0202/K00501-0802 |
| SeekOne® DD Single Cell 5’ Reverse Transcription Kit, 2 tests/8 tests | K00501-0203/K00501-0803 |
| SeekOne® DD Library Construction Kit, 2 tests/8 tests | K00202-0204/K00202-0804 |
| SeekOne® DD Single Cell Cleanup Kit, 2 tests/8 tests | K00202-0205/K00202-0805 |
Projects like TCGA and ICGC had depicted mutation patterns in various cancer tissues, but the tumour microenvironment remains unclear. Although the human tumour atlas network (HTAN), which utilizes 3′ single-cell transcriptome-seq technology, has provided valuable information on the cellular composition, it cannot detect mutations, leaving many questions and assumptions in tumour research, for example:
- Do driver mutations really occur in just tumour cells?
- If some mutations make cancer cells sensitive to certain treatments, why do some patients still have no response to those treatments?
- Tumours are the result of accumulated gene mutations. How many mutations do normal cells need to accumulate to become tumour cells, and what functional impact do these mutations have on tumour cells?
- When multiple mutations or co-mutations occur, are they in the same group of cancer cells or different ones, and how can we distinguish when considering therapeutic methods for patients?
- What is the underlying mechanism of tumour metastasis, which tumour clone contributes to the metastasis, what are their characteristics, and how can we control it?
- Is acquired resistance caused by new mutations or transcriptional reprogramming during therapy?
- In the era of precision medicine, how can we effectively select appropriate drug targets and tailor effective treatments based on the specific characteristics of individual patients?
“rceause” and phenotype is the “effect“. Detecting both mutations and expression within single scecFllAs SisT-essesqenteiaclhfnoorloagcyomcapprteuhrens sifvuell-ulnednegrtshtanRdNinAgs owf itchharllaenndgoinmg qpureimstieornss.toThdeeitnencot vbatoitvhe dmeuvtealotipomnenatnadndemxpetraesstsaisoins.,
The Single-Cell Sequencing Workflow
The single-cell sequencingworkflow includes four crucial steps: 1) initialtissue preparation, 2) single-cell
isolation and library preparation, 3) sequencingand primary analysis, and 4) data visualization and
interpretation (Figure 1). There are experimental considerations and critical steps throughout the workflow
that can impact results and determine the successof a study. Awell-planned and executed experiment is
important toensure accurate data and draw insightful conclusions.
Variousmethods have been developed forthe capture and isolation of single cells, and selection of an optimaapproach depends largely on the research question and sample type. Similarly, various techniques are
available for profilingthe genome, transcriptome, epigenome, and proteome of isolated cells, and the
method chosen willdetermine library preparation, sequencing, and downstream analyses. This chapter
discussesoptions available for single cell isolation and highlights techniques used for global characterization of
isolated cells.
Considerations for sequencing
Experiment planning
Read depth
Sequencingcoverage fortraditionalor bulk samplesdescribes the average number ofreads that align to, or
“cover,” known reference bases. NGScoverage leveloften determineswhether variant discovery can be
made with a certain degree of confidence at particular base positions. Sequencingcoverage requirements
vary by application. At higherlevelsof coverage, each base is covered by a greater number of aligned
sequence reads, or a greater“depth,” sobase calls can be made with a higher degree of confidence.16
Enrichment Sequencing
Sequencing
For Research Use Only. Not for use in diagnos tic p rocedures. 15
For various single-cell sequencingapplications, read depth isdiscussed not in the number ofreadsper base,
but in the number ofreadsper cell. The required sequencingdepth for a single-cell sequencingrun will
depend on several factors, includingsample type, the number of cells tobe analyzed, experimental
objectives, and more. For single-cellRNA-Seq, it hasbeen reported that unbiased cell-type classification
within a mixed population ofdistinct celltypes can be achieved with as few as10,000to50,000readsper
cell.17 Such lowerread depth can be practical and economical ifthe experimentalobjective is toidentify rare
cellpopulationsortoscan cells for presence ofmixed populations. However, this read depth may not be
sufficient when more homogeneous cellpopulations are studied, and it isunlikely toprovide detailed
information on gene expression within any given cell. In such casesdeeper sequencingmay be required for
improvingcell identification and detection ofgeneswith low expression. Indeed, it hasbeen reported that
500,000readsper cell are sufficient todetect most genes expressed in a cell, and 1,000,000readsper cell
approaches sequencingsaturation, enablingthe estimation ofthe mean and variance ofgene
expression.18,19 Ultimately, the required sequencingdepth will largely depend sample type and
experimentalobjective and willneed tobe optimized for each study.
Paired-end vs. single-read sequencing
Single-read sequencinginvolves sequencingDNAfrom only one end and is the simplest way toutilize Illumina
sequencing. Single-read sequencingdelivers large volumesofhigh-quality data, faster and cheaperthan
paired-end sequencing.20 Single-read runs can be a good choice for certain methods such as smallRNASeq or chromatin immunoprecipitation sequencing(ChIP-Seq). In contrast, paired-end sequencinginvolves
sequencingboth endsofDNAfragments in a library and aligningthe forward and reverse reads as read pairs.
This results in better alignment ofreads, especially across repetitive, difficult-to-sequence regions. All Illumina
NGSsystems are capable ofpaired-end sequencing.
Run QC
Percent passing filter
Percent passingfilter(%PF) is an important sequencingQCmetric that refers tothe number of clusters that
have passed a filter and willbe retained for downstream analysis. With nonpatterned flow cells, Real-Time
Analysis software evaluates clustersduringimage analysis early in the sequencingrun duringtemplate
generation. Any dim orlow-quality clusters are removed, effectively actingas a prefiltration step, resultingin
relatively high %PF values. With patterned flow cells, fixed clusterlocations eliminate the need fortemplate
generation, sothere isnoprefiltration ofunderperformingclusters. Instead, suboptimal clusters are filtered
duringthe later stage of chastity filtration. Chastity isdefined as the ratioofthe brightest base intensity divided
by the sum ofthe brightest and second brightest base intensities. Clusters “pass filter”ifnomore than one
base callhas a chastity value below 0.6in the first 25sequencingcycles. This filtration process removes the
least reliable clusters from the image analysis results. Consequently, for patterned flow cells the %PF metric
willbe lower(than for nonpatterned flow cells), but it willnot affect performance or data quality.
Data Analysis, Visualization, and Interpretation
Introduction
Afterthe single-cell sequencingrun is complete, downstream analysis can be performed. Generally, the
analysispipeline for single-cell sequencingexperiments involves three phases: primary analysis (base calling),
secondary analysis (demultiplexing, alignment, and genetic characterization), and tertiary analysis (data
visualization and interpretation) (Figure 8). There isnoone, correct way tocarry out an analysispipeline for
single-cell sequencingexperiments. Many approaches and software programs are available for each step in
the pipeline. The research objective, single-cell isolation platform, and general lab considerationswill largely
determine the specific pipeline used. This chapter outlines the steps involved in single-cell sequencing
analysis and some ofthe tools available.