Skip to Content

SeekOne ® DD Single Cell Full-lengthRNASequence Transcriptome-seq (scFAST-seq)

The SeekOne® Digital Droplet (SeekOne® DD) High-Throughput Single Cell Full-Length RNA Sequence Transcriptome-seq (scFAST-seq) Kit


For MicroRNA and viral RNA profiling of Single Cell Libraries


The scFAST-seq kit is designed to build comprehensive full-length RNA libraries from up to 12,000 cells, making it highly efficient for single-cell RNA sequencing (scRNA-seq). It utilizes unique techniques such as semi-random primers, reverse transcription, template swapping, and rRNA removal by blockers to achieve robust and comprehensive sequencing. 


scFAST is used for Single Cell and Single Nuclei 2nd generation Seq of:


  1. LncRNA seq
  2. CircRNA seq
  3. MiRNA seq
  4. ViralRNA seq


Combined with 3' scRNA seq data in one SeekOne DD library run!


Unlike traditional 3' scRNA-seq, scFAST-seq excels in capturing non-polyadenylated transcripts, offering greater transcript coverage and the ability to identify more splice junctions. Little poly(A)+ mRNAs are used add the poly(T) tract, UMI or barcode.


Enhanced Transcript Detection:

 Captures both polyA and non-polyA tailed RNA, allowing for comprehensive profiling of viral RNA transcripts and other non-coding RNA molecules.


Full-Length RNA Libraries: 

Achieves full-length transcript genome body coverage, which is ideal for studying alternative splicing and long non-coding RNA. LncRNA that translate in microRNA's.


MicroRNAs (miRNAs) profiling from small, non-coding RNA molecules that regulate gene expression by binding to messenger RNAs (mRNAs) and inhibiting their translation or promoting their degradation.

  1. Chromium
  2. SeekOne DD


 Long non-coding RNAs (lncRNAs) are a diverse class of RNA molecules that are longer than 200 nucleotides and do not code for proteins. 

Some lncRNAs can give rise to miRNAs, which play critical roles in various cellular processes. 

scFAST-seq detects miRNAs derived from lncRNAs


1. miR-675 (from H19 lncRNA)

  • Function: miR-675 is involved in regulating cell proliferation, differentiation, and apoptosis. It is associated with tumorigenesis, playing a role in cancers like gastric, colorectal, and liver cancers. The parent lncRNA, H19, is also known to be involved in growth regulation and is upregulated in various cancers.
  • Role in Disease: Tumor suppressor or oncogene depending on the context.

2. miR-7 (from LINC00472)

  • Function: miR-7 functions mainly as a tumor suppressor. It is involved in inhibiting pathways that promote cell proliferation and survival, such as the EGFR/PI3K/AKT pathway. It is also associated with neurogenesis and brain development.
  • Role in Disease: Downregulated in various cancers, including breast, lung, and brain cancers.

3. miR-21 (from GAS5 lncRNA)

  • Function: miR-21 is one of the most studied oncogenic miRNAs. It targets multiple tumor suppressor genes, promoting cell proliferation, invasion, and metastasis. The lncRNA GAS5 is known to interact with miR-21 and can regulate its expression.
  • Role in Disease: Upregulated in many cancers (e.g., breast, lung, colorectal), promoting tumor growth and survival.

4. miR-122 (from lncRNA HULC)

  • Function: miR-122 is a liver-specific miRNA that plays a crucial role in liver metabolism, lipid regulation, and hepatitis C virus (HCV) replication. It has a connection with HULC (Highly Upregulated in Liver Cancer) lncRNA, which sponges miR-122 and contributes to liver cancer progression.
  • Role in Disease: Important in liver diseases, including hepatocellular carcinoma (HCC).

5. miR-34a (associated with MALAT1 lncRNA)

  • Function: miR-34a acts as a tumor suppressor by promoting apoptosis and inhibiting cell proliferation. It is connected with MALAT1 (Metastasis-Associated Lung Adenocarcinoma Transcript 1), where it regulates the expression of genes involved in cell cycle control and metastasis.
  • Role in Disease: Downregulated in various cancers, including lung, liver, and breast cancer.

6. miR-200 family (associated with HOTAIR lncRNA)

  • Function: Members of the miR-200 family (e.g., miR-200a, miR-200b, miR-200c) are involved in the regulation of epithelial-to-mesenchymal transition (EMT), a key process in cancer metastasis. They are also connected to the lncRNA HOTAIR, which regulates gene expression related to cancer progression.
  • Role in Disease: Frequently dysregulated in cancers, playing roles in inhibiting metastasis.

7. miR-31 (from LOC554202)

  • Function: miR-31 is involved in regulating cell proliferation, invasion, and migration. It can act as both a tumor suppressor and an oncogene depending on the tissue context. The lncRNA LOC554202 is implicated in the modulation of miR-31 expression.
  • Role in Disease: Associated with various cancers, such as breast, colorectal, and lung cancer.

8. miR-155 (from BIC lncRNA)

  • Function: miR-155 is known for its roles in immune responses, inflammation, and cancer. It can regulate genes involved in immune cell differentiation and response. The lncRNA BIC (B-cell Integration Cluster) is the precursor of miR-155.
  • Role in Disease: Overexpressed in hematological malignancies and various solid tumors, contributing to inflammation and cancer.

9. miR-214 (from DNM3OS lncRNA)

  • Function: miR-214 is involved in cell differentiation, proliferation, and apoptosis. It also plays a role in cardiovascular development and cancer. The lncRNA DNM3OS acts as its host gene, contributing to various cellular processes.
  • Role in Disease: Dysregulated in conditions like cancer, cardiac hypertrophy, and fibrosis.

10. miR-143/145 cluster (from LINC00473)

  • Function: This miRNA cluster regulates smooth muscle differentiation, proliferation, and migration. The LINC00473 lncRNA is known to act as a host gene for miR-143/145, with important implications in cancer biology.
  • Role in Disease: Involved in cardiovascular diseases and acts as a tumor suppressor in various cancers.

These miRNAs measured with SeekOne scFAST seq lncRNAs play critical roles in various biological processes and diseases, including cancer, cardiovascular disorders, and immune responses. 

MicroRNAs (miRNAs) play a significant role in regulating gene expression and are implicated in various biological processes.

Dysregulation of miRNAs detected by scFAST seq are responsible for several diseases, including cancer, cardiovascular disorders, neurological diseases, and more. 


SeekOne DD scFAST seq anayses are done to detect miRNAs associated with specific diseases:


1. Cancer

miR-21: Often termed an "oncomiR," it is overexpressed in many cancers (e.g., breast, lung, colorectal, and liver cancers). It promotes tumor growth by inhibiting tumor suppressor genes, thus encouraging cell proliferation, invasion, and survival.

miR-34a: Acts as a tumor suppressor by promoting cell cycle arrest and apoptosis. It is downregulated in cancers like breast, liver, and pancreatic cancer.

miR-155: Overexpressed in hematological malignancies such as lymphoma and leukemia, as well as in solid tumors like breast and lung cancer. It plays a role in promoting inflammation and tumor growth.

miR-125b: Dysregulated in various cancers, including breast, prostate, and brain cancer. It can act as either an oncogene or tumor suppressor, depending on the context.

2. Cardiovascular Diseases

miR-1: Plays a role in cardiac function, including cardiac hypertrophy and arrhythmias. Dysregulation can lead to abnormal heart rhythms and myocardial infarction.

miR-133: Essential for cardiac muscle development and function. Its downregulation is linked to cardiac hypertrophy and heart failure.

miR-208: Associated with heart failure and hypertrophy. It regulates genes involved in cardiac muscle function and is a biomarker for heart-related conditions.

miR-145: Regulates vascular smooth muscle cell differentiation. Abnormal levels are implicated in conditions like atherosclerosis and hypertension.

3. Neurological Disorders

miR-132: Involved in neuronal development, synaptic plasticity, and neuroinflammation. Dysregulation is linked to Alzheimer's disease, schizophrenia, and epilepsy.

miR-124: Critical for neuronal differentiation and brain development. Altered expression is associated with neurodegenerative diseases like Parkinson's and Alzheimer's.

miR-9: Plays a role in neurogenesis and brain development. Dysregulation has been observed in neuropsychiatric disorders such as schizophrenia and Huntington's disease.

miR-219: Linked to myelination in the central nervous system. Altered expression is associated with multiple sclerosis and other demyelinating disorders.

4. Metabolic Disorders

miR-375: Involved in insulin secretion and glucose metabolism. Dysregulation is associated with diabetes, particularly Type 2 diabetes, as it affects pancreatic beta-cell function.

miR-103/107: Associated with insulin sensitivity. Overexpression is linked to insulin resistance and obesity.

miR-122: Plays a crucial role in lipid metabolism. It is overexpressed in non-alcoholic fatty liver disease (NAFLD) and associated with cholesterol regulation.

5. Immune-Related Diseases

miR-146a: Regulates immune response and inflammation. It is associated with autoimmune diseases such as rheumatoid arthritis, psoriasis, and lupus. Dysregulation leads to chronic inflammation and altered immune responses.

miR-155: Plays a critical role in immune cell differentiation and function. Overexpression is linked to autoimmune conditions, including multiple sclerosis and Crohn's disease.

miR-223: Involved in the regulation of inflammation and immune cell function. Dysregulation is observed in conditions such as sepsis, inflammatory bowel disease (IBD), and asthma.

6. Viral Infections

miR-122: Exploited by the Hepatitis C virus (HCV) to stabilize its RNA, aiding in viral replication. It is a target for antiviral therapies.

miR-29: Plays a role in the immune response against viral infections. Dysregulation is observed in HIV infection, contributing to immune cell dysfunction.

miR-155: Upregulated during infections with viruses like Epstein-Barr virus (EBV) and Human T-cell leukemia virus (HTLV), supporting viral replication and pathogenesis.

7. Fibrotic Diseases

miR-21: Involved in the regulation of fibrosis by promoting fibroblast activation and collagen deposition. Dysregulated in conditions like pulmonary fibrosis, liver fibrosis, and renal fibrosis.

miR-29: Functions as an anti-fibrotic miRNA by inhibiting collagen production. Its downregulation is associated with fibrotic diseases such as liver cirrhosis and cardiac fibrosis.

miR-192: Contributes to kidney fibrosis by regulating genes involved in extracellular matrix production and cell proliferation.

8. Muscular Disorders

miR-206: Involved in muscle regeneration and differentiation. Dysregulated expression is associated with muscular dystrophies and other neuromuscular diseases.

miR-1 and miR-133: Play critical roles in muscle development. Altered levels are associated with conditions such as cardiac hypertrophy and myopathies.

These examples illustrate the significant roles that miRNAs play in various diseases, acting as either oncogenes, tumor suppressors, or regulators of metabolic, immune, and developmental processes. Their ability to control multiple genes makes them potential targets for therapeutic interventions and biomarkers for diagnosis and prognosis.


Mutation and Gene Expression Profiling: 


Combines the detection of somatic mutations with gene expression at the single-cell level, aiding in understanding tumor heterogeneity and precision medicine.


Target Region Enrichment:


 Capable of detecting specific mutations and cell states, offering valuable insights for cancer research.

One-Stop Solution: When used with the SeekOne® Digital Droplet System (SeekOne® DD) and SeekSoul Tools software, it provides a streamlined solution from nucleic acid labeling to data analysis.

Applications

Tumor Heterogeneity: Enables detailed studies of mutation profiles and gene expression across individual tumor cells.

Isoform Analysis: Facilitates research on alternative splicing and non-coding RNA involved in gene regulation.

Viral Research: Detects viral RNA transcripts, allowing for in-depth analysis of virus-host interactions and microenvironmental changes during infection.


Research & Development: 


Supports drug development, clinical evaluation, and manufacturing quality control by providing a comprehensive view of cellular states.

Multi-Omics Integration: Overcomes the limitation of traditional single-omics approaches by integrating multiple data dimensions for a more holistic understanding of cellular biology.

The scFAST-seq kit offers a robust and versatile platform for researchers focused on oncology, virology, and transcriptomics, providing an all-in-one solution for high-resolution single-cell analysis.


scFAST seq also picks up Circular RNAs (circRNAs) 


These non-coding RNAs characterized by their covalently closed loop structure, which makes them more stable than linear RNAs. They play important roles in gene regulation, often acting as microRNA (miRNA) sponges, regulating transcription, or even translating into proteins. Here are some of the most well-studied and important circRNAs, along with their functions:


1. ciRS-7 (CDR1as)

Function: ciRS-7, also known as CDR1as (cerebellar degeneration-related protein 1 antisense), is one of the best-known circRNAs. It acts as a sponge for miR-7, binding to multiple miR-7 molecules and inhibiting their activity. miR-7 is involved in regulating various genes related to cancer, neurodevelopment, and insulin secretion.

Role in Disease: Dysregulation of ciRS-7 has been associated with cancer progression (e.g., colorectal, liver, and breast cancer), as well as neurological disorders such as Alzheimer's disease.

2. circHIPK3

Function: Derived from the HIPK3 gene, circHIPK3 acts as a sponge for various miRNAs, including miR-124, miR-558, and others, influencing cell proliferation, migration, and angiogenesis. It is known for its role in regulating pathways associated with cancer cell growth and metabolism.

Role in Disease: Overexpression of circHIPK3 has been observed in multiple cancers, including bladder, colorectal, and liver cancer, where it promotes tumor growth and survival.

3. circFOXO3

Function: circFOXO3 is derived from the FOXO3 gene and is known to regulate cell cycle progression and apoptosis. It can interact with proteins such as p21 and CDK2, preventing them from promoting cell cycle progression, thereby acting as a tumor suppressor.

Role in Disease: It has been implicated in the regulation of cancer cell proliferation, particularly in breast cancer and cardiovascular diseases, where it is thought to protect against stress-induced cell damage.

4. circZNF91

Function: circZNF91 can act as a sponge for miR-23b-3p, which is involved in regulating immune responses, cell proliferation, and apoptosis. It affects the expression of various genes by modulating miRNA activity.

Role in Disease: Dysregulation of circZNF91 is linked to tumorigenesis and cancer progression, especially in liver and breast cancer.

5. circMTO1

Function: circMTO1 is known to inhibit the function of miR-9, a microRNA that promotes tumor growth. By sponging miR-9, circMTO1 acts as a tumor suppressor, promoting the expression of target genes that inhibit cancer cell proliferation.

Role in Disease: circMTO1 is downregulated in hepatocellular carcinoma (HCC) and acts as a tumor suppressor, with potential applications as a biomarker for HCC prognosis.

6. circITCH

Function: Derived from the ITCH gene, circITCH acts as a sponge for miR-7, miR-17, and miR-214, which are associated with pathways involved in cell proliferation and apoptosis. It promotes the expression of ITCH, a ubiquitin-protein ligase that plays a role in tumor suppression.

Role in Disease: circITCH is associated with the suppression of tumorigenesis in cancers such as colorectal, lung, and bladder cancer, making it a potential biomarker and therapeutic target.

7. circCCDC66

Function: circCCDC66 is known to regulate genes involved in cell proliferation, migration, and invasion. It acts by sponging miRNAs such as miR-33b and miR-93, affecting cancer-related signaling pathways.

Role in Disease: It has been shown to promote tumor growth in colorectal cancer and is associated with cancer progression, particularly in gastrointestinal malignancies.

8. circRNA_100290

Function: This circRNA has been shown to regulate miR-29 and is involved in modulating cell proliferation, apoptosis, and migration. It is also associated with the regulation of cancer-related genes.

Role in Disease: CircRNA_100290 is upregulated in oral squamous cell carcinoma and promotes cancer cell growth, making it a candidate for diagnostic markers in this type of cancer.

9. circPVT1

Function: circPVT1 is derived from the PVT1 gene locus and functions as a miRNA sponge, particularly for miR-125, miR-3666, and others. It influences cancer cell proliferation and survival by affecting key oncogenic pathways.

Role in Disease: circPVT1 is overexpressed in various cancers, including gastric, lung, and breast cancers, and is associated with poor prognosis. It plays a role in tumor growth and resistance to chemotherapy.

10. circCDR1

Function: circCDR1 (also known as circRNA sponge for miR-7 or CDR1as) acts as a sponge for miR-7, modulating its effects on gene regulation. This circRNA is highly abundant in brain tissues.


We need to used scFAST seq to study these Diseases: 

Linked to neurological disorders, such as Alzheimer's disease, and also associated with cancers where miR-7 plays a key regulatory role.


SeekOne DD scFAST seq contributes to circRNAs profiling as important players in cellular regulation, and their roles in diseases, especially cancer and neurological disorders, have made them targets for diagnostic and therapeutic development. 

Their ability to modulate miRNAs and other gene expression pathways gives them significant importance for the Single Cell and Single Nuclei analysis of these mentioned diseases. 


SeekOne DD scFAST seq is also used to detect Non-polyadenylated viral RNAs

 RNA molecules produced by certain viruses that do not have the poly(A) tail are a common feature found at the 3' end of eukaryotic mRNAs. 

Unlike most eukaryotic mRNAs that are polyadenylated to generate mRNA isoforms that differ only in their 3′ UTRs , some viruses produce RNAs without this polyA tail, which influences how they are processed, translated, and stabilized within host cells. Here are some notable examples:

1. Influenza Virus

  • RNA Type: Negative-sense segmented RNA genome.
  • Description: Influenza viruses (A, B, and C) have an RNA genome that does not possess poly(A) tails. Instead, the viral RNA segments use host cell mRNA as primers (a process called "cap-snatching") to initiate viral mRNA synthesis. The absence of a poly(A) tail does not prevent the influenza virus from efficiently translating its mRNAs because it uses other mechanisms for mRNA stability and translation.

2. Reoviruses (e.g., Rotavirus)

  • RNA Type: Double-stranded RNA (dsRNA).
  • Description: Reoviruses, including rotaviruses, have segmented double-stranded RNA genomes. Their mRNAs are non-polyadenylated but still serve as templates for translation. These viruses produce mRNAs that are capped but lack the poly(A) tails, using alternative strategies to stabilize their transcripts and promote efficient translation.

3. Hantaviruses

  • RNA Type: Negative-sense single-stranded RNA (ssRNA).
  • Description: Hantaviruses are part of the Bunyaviridae family, which produces non-polyadenylated viral mRNAs. These viruses use a similar cap-snatching mechanism as influenza to initiate transcription and often stabilize their RNA transcripts without the need for polyadenylation.

4. Rabies Virus (Rhabdovirus Family)

  • RNA Type: Negative-sense single-stranded RNA (ssRNA).
  • Description: Rabies virus produces non-polyadenylated viral RNAs. Its RNA-dependent RNA polymerase (RdRp) transcribes the genome into mRNAs that are not polyadenylated but have a different structure to support efficient translation within host cells.

5. Hepatitis Delta Virus (HDV)

  • RNA Type: Circular, negative-sense single-stranded RNA (ssRNA).
  • Description: HDV produces small, non-polyadenylated RNA molecules that rely on host RNA polymerase II for replication. Despite lacking a poly(A) tail, HDV RNA can effectively replicate and be packaged within the host cell by using other structural features and mechanisms.

6. Bornavirus

  • RNA Type: Negative-sense single-stranded RNA (ssRNA).
  • Description: Bornaviruses produce non-polyadenylated mRNAs. These viruses can establish persistent infections in the nervous systems of their hosts, and their RNA transcripts are processed and translated without a poly(A) tail.

7. Flaviviruses (e.g., Dengue Virus, West Nile Virus)

  • RNA Type: Positive-sense single-stranded RNA (ssRNA).
  • Description: While most Flaviviruses, like dengue and Zika, have polyadenylated tails, certain members of the family produce RNA with non-polyadenylated 3' ends. These viruses utilize RNA secondary structures to maintain the stability of their genome and facilitate translation.

8. Arenaviruses (e.g., Lassa Virus)

  • RNA Type: Negative-sense or ambisense single-stranded RNA (ssRNA).
  • Description: Arenaviruses generate non-polyadenylated mRNAs. The genome consists of two segments (L and S), and the viruses use a unique transcription strategy that allows them to produce functional RNAs without needing polyadenylation.

9. Ebola Virus (Filoviridae Family)

  • RNA Type: Negative-sense single-stranded RNA (ssRNA).
  • Description: Ebola virus produces non-polyadenylated mRNAs, and instead of polyadenylation, it uses transcriptional strategies to stabilize and translate its RNA. The viral genome encodes an RdRp that processes the viral RNAs, which remain non-polyadenylated but are still functional.

10. Nodaviruses (e.g., Flock House Virus)

  • RNA Type: Positive-sense single-stranded RNA (ssRNA).
  • Description: Nodaviruses produce genomic and subgenomic RNAs that are non-polyadenylated. These viruses are known to infect insects and can use other structural elements within their RNA to ensure stability and efficient translation.

Mechanisms of Stability Without Poly(A) Tails:

While most eukaryotic mRNAs rely on poly(A) tails for stability and translation, non-polyadenylated viral RNAs utilize alternative mechanisms, including:

  1. RNA Cap Structures: Enhancing stability and translation initiation.
  2. RNA Secondary Structures: Stem-loop formations and other structures that prevent degradation.
  3. Viral Proteins: Binding to viral RNA to stabilize and facilitate translation.
  4. Cap-snatching Mechanism: Using fragments of host mRNAs to ensure the viral RNA is recognized and efficiently translated.

These examples highlight the diversity viral RNA's detected by the SeekOne DD scFAST seq pleudo long read 2nd generation NGS libraries, showing how different viruses have adapted their genome structures and transcription methods to thrive even without the typical eukaryotic poly(A) tail.


Author:

Lieven Gevaert, 

Gentaur Laboratories Leuven, Belgium