Is third-generation sequencing/long-read-sequencing the only way to identify the full length sequence of a novel transcript?

Question

[wangziwei0010]

Thanks for your attention,

TLDR: How to get the full length sequence of a novel transcript?

Details:

1. There is a novel transcript A I identified using short-read sequencing, through assembly. It contains an unique sequence I have validate using PCR, I refer to it as ATCGATCG for illustration.
2. Because In-silico assembly using short, pair-end read is quite noisy. I want to clearly know the full length sequence of the transcript A, which is XXXXATCGATCGXXXXXXXX where X indicate bases I didn't know.
3. I know that 3rd generation sequencing could get full length transcript. However, it's expensive. And I'm not interested in every transcript, transcript A.
4. So, Is there any cost-effective way to get the full length sequence?

Any clue or guidance is highly appreciated!

Answer 1

[Mensur Dlakic]

Doesn't seem like a bioinformatics question.

As detailed as your question is, it misses a crucial information of the type of organism you are dealing with. I am going to assume it is a eukaryote, as prokaryotic transcripts are usually easier to define.

I suggest you ask around for a cDNA library kit, or do a Google search for it.

• https://www.thermofisher.com/us/en/home/technical-resources/technical-reference-library/pcr-cdna-synthesis-support-center/cdna-libraries-construction-support.html
• https://www.takarabio.com/products/mrna-and-cdna-synthesis/cdna-synthesis-kits
• https://store.nanoporetech.com/us/direct-cdna-sequencing-kit.html

After creating a cDNA library, you find the desired clone by hybridization and sequence only that plasmid.

[wangziwei0010]

Thank you for your time and valuable suggestions!