SeqInsight is offering Iso-Seq service by producing full-length transcripts without assembly. Strict quality control following every procedure is executed to ensure comprehensive and accurate results.

The Introduction of Iso-Seq

In eukaryotic organisms, a single gene may encode a surprising number of proteins after alternatively spliced, each with a distinct biological function. There are known human genes that have very different functions depending on which splice variant is expressed. With alternative splicing so critical to genome function.

Short-read RNA-Seq works by physically shearing transcript isoforms into smaller pieces and reassembling, leaving possibilities for mis-assembly or incomplete capture of the full diversity of isoforms from genes of interest.  It’s important to capture full-length transcripts. By taking advantage of PacBio SMRT long reads sequencing technology, the isoform sequencing (Iso-Seq) can easily cover complete transcript from the 5′ end to the 3′-poly A tail without the need of fragmentation to obtain full-length cDNA sequences, which is useful to identify new transcripts and new introns, thus accurately identifying isoforms, alternative splicing sites, fusion gene expression, and allelic expression.

Comparing with Illumina platform, PacBio analysis can easily detect very long polycistronic RNA molecules (called complex transcripts), and reveal a wide variety of novel transcriptional overlaps between adjacent and distant genes situated in parallel. It is helpful to raise the possibility to study a genome-wide network exerting joint control on gene expression and replication.

Project Workflow

Sample Requirements:

• RNA amount: Total RNA ≥ 5 ug (without degradation or DNA contamination)
• RNA purity: OD260/280 = 1.8~2.2; OD260/230 ≥ 1.5
• RNA quality: 28S:18S ≥ 1.5，RIN ≥ 7

Sequencing Strategy:

PacBio Platforms, Library type > 4K, 6~10 G/per sample

Data Analysis

Transcriptome with no Reference
Standard data analysis

• Full-length Isoform analysis, including Full-length Isoform correction, Classification, Reduced Redundancy
• Transcriptome annotation, including Gene Ontology, KEGG pathway, KOG or COG, Swissport
• Gene Structures analysis, including alternative splicing, LncRNA, SSR, CDS

Advanced data analysis

• Map to Reference
• Gene expression level analysis
• Differentially expressed gene analysis
• Differentially expressed transcriptome KEGG enrichment analysis
• Differentially expressed gene heatmap
• … (more upon request)

Transcriptome with Reference
Standard data analysis

• Full-length Isoform analysis, including Reference Genome mapping, Full-length Isoform correction, Classification, Reduced Redundancy
• Transcriptome annotation, including Gene Ontology, KEGG pathway, KOG or COG, Swissport
• Gene Structures analysis, including alternative splicing, LncRNA, SSR, CDS, novel transcriptome prediction, Identification of fusion gene

Advanced data analysis

• Map to Reference
• Gene expression level analysis
• Differentially expressed gene analysis
• Differentially expressed transcriptome KEGG enrichment analysis.
• Differentially expressed gene heatmap
• … (more upon request)

Analysis pipelineis