ISO-Seq/PacBio Transcriptome Sequencing
Description
“Isoform Sequencing” (Iso-seq) developed by Pacific Biosciences (PacBio), is based on long-read sequencing technology and allows researchers to identify new isoforms with extraordinary precision.
RNA sequencing has become the most frequently used method for the majority of researchers conducting gene expression profiling. However, it is difficult to obtain a complete picture of the transcriptome because short reads cannot accurately assemble complex transcripts.
Isoform Sequencing (Iso-seq) developed by Pacific Biosciences (PacBio), is based on long-read sequencing technology. The unique long-read sequencing feature allows this method to identify new isoforms with extraordinary precision. The Iso-Seq application generates full-length cDNA sequences — from the 5’ end of transcripts to the poly-A tail — eliminating the need for transcriptome reconstruction using isoform-inference algorithms. The Iso-Seq method provides accurate information about alternatively spliced exons and transcriptional start sites. It also reveals information about poly-adenylation sites for transcripts across the full complement of isoforms within targeted genes or the entire transcriptome.
Key Service Details
Iso-Seq/PacBio transcriptome sequencing services are executed on the Sequel I/II platform.
Reports and output data files are delivered in industry standard file formats: BAM, .xls, .png and FASTQ data.
Sequencing Standards
20Gb sequencing data per sample is recommended.
Data Analysis
- Clean data, standard and customized data analysis options available
- Data storage services and bioinformatics applications available upon request
Sample Requirements
Recommended:
Mass ≥ 3 µg
Concentration ≥ 285ng/µL
RIN ≥7.5
28S/18S(23S/16S) ≥1.2
Required:
1.5µg ≤ m < 3 µg
RIN ≥7.5
28S/18S(23S/16S) ≥1.2
Turnaround Time
Typical 40 working days from sample QC acceptance to sequencing data availability
Highlights of Iso-Seq Service
1 ）Absolute Quantification with UMI Technology.
We can process your total RNA, whole blood, cell line, fresh frozen tissue samples, with the following general requirements: Each original transcript is marked by a unique molecular identifier (UMI), which includes 6-8 random bases. Counting the copy number of transcripts with a UMI based approach enables accurate quantification of the isoform without the interference of sequencing duplication. The UMI technology is built-in multi-throughput Iso-Seq and polyA ISO-Seq workflows. Both the UMI technology and the multi-insert sequential ligation technique are BGI patent-protected.
2 ）Greater Transcripts Detection with Multi-throughput Iso-Seq
Compared with standard Iso-Seq, Multi-throughput Iso-Seq can obtain 3-5 times more effective reads and allow users to detect double amount of transcripts with the same volume of sequencing data.
Notes: Total sequencing data amount as follows: UHRR-9.47 Gb (Standard Iso-Seq); 9.57 Gb (Multi-throughput Iso-Seq); Maize-11.04 Gb (Standard Iso-Seq); 19.3 Gb (Multi-throughput Iso-Seq). Polymerase-reads: The number of polymerases generated high quality reads. Polymerase reads will be then trimmed to preserve only the high-quality region, which includes bases from adaptors and single or multiple passes around a circular template. Effective reads: Each cDNA template molecule is considered as an “insert” and each pass through the insert is called a effective read. A polymerase read made by Multi-throughput Iso-Seq can contain more than one unique insert.
3 ）PolyA length analysis with polyA Iso-Seq
A variety of studies have reported the importance of polyA tail length for gene expression activity. The polyA Iso-Seq service provides additional information of polyA sequence, such as the length distribution of polyA; the occurrence frequency of other bases in polyA and the correlation between polyA length and gene expression.
