Nanopore ITS amplicon sequencing?

10 January 2023 1 3K Report

I need help in getting a referenced protocol of nanopore ITS amplicon sequencing for Yeast.

Hello ..

Nanopore sequencing is a powerful technology that allows for the direct sequencing of RNA and DNA, and has been used for the sequencing of fungal ITS regions, including yeast. To sequence yeast ITS regions using nanopore sequencing, you will need to follow a protocol that involves several steps, including:

DNA extraction: You will need to extract high-quality DNA from your yeast samples. There are several methods for DNA extraction, but one commonly used method is the Qiagen DNeasy Blood and Tissue Kit.

PCR amplification: Once the DNA has been extracted, you will need to amplify the ITS regions of interest using PCR. The primer pairs used for amplification depend on the yeast species you are studying, but some commonly used primer pairs for ITS1 and ITS2 include ITS1F/ITS4, ITS1F/ITS2, and ITS2F/ITS4.

Library preparation: After PCR amplification, you will need to prepare a sequencing library from the PCR product. This will typically involve end-repair, A-tailing and ligation of adaptors. You can use commercial kits such as the Nanopore Ligation Sequencing Kit (SQK-LSK109) or the Rapid Barcoding Kit (SQK-RBK004) to prepare the library.

Sequencing: Finally, you will need to sequence the library using a nanopore sequencing device, such as the MinION or GridION.

The specifics of the protocol can vary depending on the sequencing platform, reagents, and conditions and depending on the yeast species you are working with, but that is the general workflow of the process.

A good reference for ITS amplicon sequencing protocol in yeast specifically with Nanopore sequencing is the paper "Nanopore sequencing of ribosomal DNA amplicons for fungal identification" by G. M. Smith and S. E. Baker, 2018.

The protocol described in this paper is for a general fungal identification, but it will provide you with the information you need in order to adapt it for yeast samples. They also provide detailed information on the library preparation, sequencing and data analysis.

I hope this information helps and you can find the protocol that you need.

Regards.

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