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Cell Biology JoVE (Open Access) Citable · DOI

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

DOI: 10.3791/59377-v
What you'll learn
  • ✓ Perform mRNA amplification from whole blood samples using nanopore-compatible protocols
  • ✓ Prepare sequencing libraries and validate flow cell quality for nanopore runs
  • ✓ Load and execute nanopore sequencing runs in resource-limited settings
  • ✓ Analyze and interpret mRNA sequencing data from whole blood samples
Protocol

Biopharma Insights Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

Difficulty
advanced
Total time
~4-6 hours per sample (including library prep and sequencing run)
Biosafety
BSL-2

Steps

1
Amplify NPC1 open reading frame from blood

Perform targeted amplification of the NPC1 ORF from whole blood mRNA samples using nanopore-compatible PCR protocols to generate sufficient material for sequencing.

▶ 00:54
2
Prepare nanopore-compatible sequencing library

Convert amplified mRNA products into sequencing-ready libraries following nanopore library preparation workflows, including end-repair and adapter ligation steps.

▶ 03:36
3
Validate flow cell functionality and integrity

Perform quality control checks on the nanopore flow cell to ensure proper pore function and absence of defects before loading samples.

▶ 05:48
4
Load samples and initiate sequencing run

Load prepared library onto the validated flow cell and start the nanopore sequencing instrument to begin mRNA data acquisition.

▶ 06:31
5
Analyze sequenced mRNA data outputs

Process and interpret nanopore raw sequencing data to assess mRNA abundance, quality metrics, and sequence accuracy from whole blood samples.

▶ 08:59
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