Digital RNA Sequencing (UMI RNA Sequencing)

Digital RNA-Seq (or UMI-RNA-Seq) is a next-generation sequencing (NGS)-based method that eliminates sequence-dependent PCR bias by barcoding RNA molecules prior to amplification. Digital RNA-Seq can not only realize the transcriptomic sequencing as conventional RNA-Seq, but also reduce the biases and errors in sequencing.

Overview

In a digital RNA sequencing assay, a unique molecular identifier (UMI) or as so-called unique identifier (UID) is added to each cDNA before library amplification. The UMI accompany the whole process of amplification, sequencing, and analysis, which contributes to the traceability during the whole process. By combining fragments from the same source (with the same sequence and UMI), the PCR amplification repeats can be accurately removed and the original state of the sample before amplification can be accurately restored. In this process, errors in PCR amplification and sequencing can also be corrected by comparing the similarities of the sequences tagged with the same UMI.

Although Conventional RNA-Seq can reflect the expression level to a certain extent, deviations or even bias between the sequencing result and the true value still exist. According to the quantitative analysis based on UMI, Digital RNA-Seq can accurately distinguish the copies generated by amplification or from the original sample, which guarantees a result close to the true value. In addition, the promising error correction function of Digital RNA-Seq significantly reduces the errors induced by amplification and sequencing procedure, allowing for more precise sequencing and deeper transcriptomics research.

UMI & Quantitative Transcriptome Sequencing

At CD Genomics, we use advanced UMI (Unique Molecular Identifier) technology to address challenges in PCR library construction that can distort library fragment abundance. Our absolute quantitative transcriptome sequencing ensures accuracy by adding a UMI to each retrotranscribed cDNA before library amplification. Through precise UMI-based data filtering, we restore each sample's initial state accurately.

Our UMI RNA sequencing results show strong agreement with Q-PCR, proving our method's reliability, especially in detecting low-abundance gene expression. After data analysis, our approach perfectly restores the original ratio of different RNA molecules, achieving "absolute quantification".

CD Genomics absolute quantitative sequencing service is designed for lower sample sizes, providing highly accurate results. Our bioinformatic analysis distinguishes between natural and amplified repeats, accurately identifying alternative splicing.

For other sample types, you can refer to our Sample Submission Guidelines or consult our technical team for information. They will be able to provide guidance and answer any specific questions you may have.

UMI & Targeted RNA Sequencing

UMI (Unique Molecular Identifier) is crucial for accurate Targeted RNA Sequencing. In this method, we create libraries from specific transcript regions.

CD Genomics specializes in targeted sequencing, especially for assessing rare variants with mutations linked to diseases, focusing on applications in cancer and oncology. Identifying true mutation sites in the original RNA faces challenges due to potential errors in the sequencing workflow. UMI helps by distinguishing errors during sequencing from mutations in the original molecule.

With UMI, variants or mutations are considered "real" when consistent across reads with the same UMI, helping rule out systematic errors during reverse transcription. CD Genomics offers Targeted RNA Sequencing with UMI technology for precise mutation detection and analysis.

UMI & Single-Cell RNA Sequencing

Experience single-cell transcriptome sequencing using Unique Molecular Identifiers (UMIs). In this approach, cells are defined by barcodes, and transcripts are tagged with UMIs.

Our solution addresses challenges like amplification bias and technical variations. UMIs, integrated at crucial stages, reduce errors in gene expression quantification. By randomly attaching UMIs to mRNAs, we eliminate PCR bias and boost the accuracy of sequencing data.

UMIs become the key unit in molecular counting, aligning with established methods in bulk transcriptome analysis. Evaluate experimental quality with the median number of UMI counts for cell-associated barcodes, providing insights into sequencing sample integrity.

Streamline your single-cell RNA sequencing workflow with UMIs for enhanced precision, accuracy, and efficient experiment evaluation.

Project Workflow

Bioinformatics Analysis Pipeline

In-depth data analysis:

• Gene fusion detection
• Differential expression analysis
• Pathway analysis
• SNV & InDel detection
• Splicing analysis
• Prediction of novel miRNAs, lncRNAs and circRNAs
• Discovery of novel transcripts and mutations, even in FFPE tissue

Sample Requirements

Cell sample ≥ 50,000 cells;
RNA sample (concentration ≥ 50 ng/µL, quantity ≥ 1 µg)
1.8 ≤ OD_260/280 ≤ 2.2, OD_260/230 ≥ 2.0, RIN ≥ 6.5, 28S:18S ≥ 1.0.
Please make sure that the RNA is not degraded nor contaminated.

Sample storage: RNA can be dissolved in ethanol or RNA-free ultra-pure water and stored at -80°C. RNA should avoid repeated freezing and thawing.

Shipping Method: When shipping RNA samples, the RNA sample is stored in a 1.5 mL Eppendorf tube, sealed with sealing film. Shipments are generally recommended to contain 5-10 pounds of dry ice per 24 hours.

Deliverable: FastQ, BAM, coverage summary, QC report, full statistical analysis & alignments, custom analysis reports on customer request.

FAQ

• • How does digital RNA sequencing eliminate amplification bias?

    • In the digital RNA sequencing (UMI RNA-Seq) process, each first-strand cDNA molecule undergoes a unique molecular tagging before being subjected to multiplex PCR using universal primer pairs. This critical step effectively addresses PCR-induced amplification bias and eliminates library construction preferences.

• • Can UMI sequencing only be used for transcript/gene quantification?

    • A: No, UMI sequencing is versatile and can be employed beyond transcript/gene quantification. Even without an amplification step, analyzing UMIs provides a convenient method to detect and calculate the abundance of individual molecules within a complex sample. This makes UMIs suitable for various sequencing methods, including the analysis of viruses, proteins, ChIP-Seq, karyotyping, and more. It extends to scenarios where determining copies by comparing coordinates is not feasible or when precise quantification is a requirement.

References:

1. Melsted P, Ntranos V, Pachter L. The barcode, UMI, set format and BUStools. Bioinformatics. 2019;35(21):4472-3.
2. Shiroguchi K, Jia TZ, Sims PA, Xie XS. Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes. Proceedings of the National Academy of Sciences. 2012;109(4):1347-52.