SMRT Full-Length RNA Sequencing

SMRT full-length RNA-sequencing is a third generation sequencing (TGS)-based method to amplify and sequence the whole transcriptome of cells or tissues. We provide SMRT full-length RNA-sequencing to help you identify new transcripts and alternative splicing events, resolve structural variants and repetitive regions.

Overview

SMRT full-length RNA-sequencing is one kind of long-read sequencing, emerged in 2011, provided by Pacific Biosciences (PacBio). SMRT is short of single molecule real-time, thus allowing you to distinguish alternative transcript isoforms at the single-cell level. SMRT full-length RNA-sequencing based on sequencing by synthesis. The RNA sample was reverse-transcription to cDNA, as DNA polymerase incorporated labeled dNTPs into the single-stranded cDNA template, the fluorescence pulses were recorded. There are two sequencing modes: circular consensus sequencing (CCS) and continuous long read (CLR) sequencing. The CCS mode is more commonly used, with highly accuracy, the average read is 13.5 kb. CLR mode allows >50 kb reads. Advantages of SMRT full-length RNA-sequencing include: 1) no assembly required with less errors in repetitive sequences; 2) capture cell-to-cell heterogeneity and regions between alleles; 3) deliver a more complete view of genetic diversity.

We provide SMRT full-length RNA-sequencing services for identifying new genes, transcripts, and alternative splicing events, resolving structural variants and repetitive regions. SMRT full-length RNA-sequencing increases the accuracy of RNA-seq at the isoform-level resolution, it allows a high--resolution view of full-length cDNA sequences without assembly.

Features

High throughput Single-Cell Insights High Resolution Multiple Applications
This method allow long reads for accurate analysis with a short sequencing time. Quantitative analysis down to single-cell levels for input samples. Discovery of more cellular differences based on high resolution analysis. Identify new genes, transcripts and alternative splicing events.

Project Workflow

Sample Preparation

1. Sample Preparation

RNA purification; quality assessment and quantification.

Library Preparation

2. Library Preparation

Single-cell cDNA enrichment; cDNA library preparation.

Sequencing

3. Sequencing

Circular consensus sequencing (CCS); continuous long read (CLR) sequencing.

Data Analysis

4. Data Analysis

Visualize and preprocess results, and perform custom bioinformatics analysis.

 
SMRT Full-Length RNA Sequencing

Data Analysis Workflow

  1. Identification of new genes
  2. Identification of new transcripts
  3. Alternative splicing events comparation between species
  4. Assign alternative isoforms to correct cell type

Sample Requirements

RNA sample (quantity ≥ 100 pg), 1.8 ≤ OD260/280 ≤ 2.2, OD260/230≥2.0, RIN ≥ 6.5, 28S:18S≥1.0. Please make sure that RNA is not significantly degraded.

Sample Storage: The sample should be stored at -80°C. Avoid repeated freezing and thawing.

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

Deliverable: FastQ, raw data, coverage summary, QC report, experiment results, custom bioinformatics analysis.

References:

  1. Xiao T, Zhou W. The third generation sequencing: the advanced approach to genetic diseases. Translational Pediatrics, 2020, 9(2): 163–173.
  2. Edi J, et al. Real-time DNA sequencing from single polymerase molecules. Science, 2009, 323(5910): 133-8.
* For Research Use Only. Not for use in diagnostic procedures.


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