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Library Preparation Guide

This guide provides detailed instructions for preparing libraries for analysis with ROBIN.

Overview

ROBIN is designed to work with libraries prepared using the Oxford Nanopore Technologies (ONT) sequencing platform.

For CNS tumor analysis, you can use either:

  1. The ROBIN-specific library preparation protocol
  2. The standard ONT LSK114 ligation protocol

Input DNA

As ROBIN uses adaptive sampling, we aim for read lengths in the range of 8-12 kb.

Library Preparation Options

Option 1: ROBIN Protocol

The ROBIN-specific library preparation protocol is detailed in our protocols.io publication: ROBIN: A Unified Nanopore-based Sequencing Assay

This protocol has been optimized for CNS tumor analysis and includes:

  • Specific fragmentation conditions
  • Optimized adapter ligation steps
  • Quality control checkpoints
  • Detailed troubleshooting guidance

Option 2: Standard ONT Protocol

Alternatively, you can use the standard ONT LSK114 ligation protocol, which is available from: - ONT LSK114 Protocol Documentation

DNA Input Requirements

  • Input DNA should be of high quality (A260/A280 ratio between 1.8-2.0)
  • Minimum input: 100ng
  • Optimal input: 500ng-1μg
  • DNA should be in TE buffer or nuclease-free water

Next Steps

After library preparation:

  1. Proceed to sequencing setup and configure MinKNOW for your run

  2. Start ROBIN analysis as described in the ROBIN Quickstart

Additional Resources