Illumina Stranded mRNA Prep Ligation FAQs

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  • Sequencing


  • Read 1 sequences will be identical to the antisense strand of DNA, which is also the first strand cDNA (only the first strand cDNA is amplified and sequenced).

    This kit will mainly target mRNA and long non-coding RNAs. Small RNAs will not be captured. 

    The read length and format (single read versus paired-end) are important considerations in the design of RNA sequencing experiments. The table below provides guidance based on internal and external data, but do not represent strict cut-offs. Needs for individual projects may vary based on multiple variables as well as user preference. Literature needs to be consulted for the most up to date resources.

    Application

    Read Type

    Read Depth per sample (mRNA/Total RNA)

    Gene profiling (gene-level counts)

    1 x 51

    >5 M / >10 M

    Differential gene expression

    2 x 76

    30–60 million

    Discovery (alternative transcripts, gene fusions, etc.)

    2 x 76

    ≥50 M / >100 M

    Complete transcriptome annotation

    2 x 76-101

    ≥100 M / ≥200 M

  • Analysis


    • DRAGEN RNA Pipeline- Performs fusion analysis and RNA quantification.
    • RNA-Seq Alignment- Aligns RNA-seq reads. Quantifies gene expression, calls small variants and gene fusions. Input for differential expression app.
    • Cufflinks Assembly & DE-Quickly assess novel transcript isoforms and gene expression levels from RNA-Seq Alignment results.