Question: Can I use amplified full-length cDNA as input into the Targeted Gene Expression assay?
Answer: We do not support the use of the Targeted Gene Expression assay with amplified full-length cDNA.
The Targeted Gene Expression assay is only supported for use with final libraries generated using the Chromium Single Cell 3’ or 5’ Gene Expression or Visium Spatial Gene Expression for fresh-frozen tissues solutions.
However, since our bait design algorithm for the Targeted Gene Expression assay produces baits that tile the entire transcriptomic space, these baits can, in theory, also be used to enrich sequences of interest from amplified full-length cDNA.
If a customer were to attempt this un-supported application, there are a number of factors to consider:
- The Universal Blockers (PN-2000290), which are a key component in preventing daisy-chain hybridization via sequencing adapters, are optimized for use with final libraries. Amplified full-length cDNA molecules are flanked by different adapter sequences than final libraries.
- The amount of library input has been optimized for use with final libraries, 300ng on average per library. Amplified full-length cDNA yields may be below such an amount.
- The Targeted Gene Expression assay allows for combining multiple libraries into a pulldown reaction because each is indexed uniquely during library construction. Amplified full-length cDNA is not indexed uniquely, therefore, such multiplexing capability would be lost.
- The post-capture PCR amplification protocol has been optimized for use with final libraries. Input amount, primer, and cycling conditions will vary when using amplified full-length cDNA.
- Full-length cDNA may contain a larger proportion of intronic sequences (from un-spliced transcripts) that can interfere with the assay.
- If desired, library construction for Illumina sequencing would still have to be completed after successful enrichment of amplified full-length cDNA.
- Full length cDNA generated from select 10x assays is compatible (but unsupported by 10x Genomics) with long read sequencing from Pacific Biosciences and Oxford Nanopore Technologies. However, enriched cDNA from the Targeted assay has not been validated by 10x Genomics, PacBio or ONT for compatibility with long read sequencing. Therefore this would be performed at the customer’s own risk. Please see the following article for information regarding which assays are compatible with each long read sequencing technology: Which 10x Assays are compatible with long-read sequencing applications?
- If interested in using long read sequencing, please reach out to either PacBio or ONT Support for additional information.
- For an example of Oxford Nanopore Technologies' long-read sequencing on 10x Genomics cDNA that has been enriched for BCR and TCR cDNA, please refer to Singh et al 2019.
Products: Targeted Gene Expression