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Amplicon Sequencing using the Long-Read Sequencing Technologies
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Methodological Comparison of Short-Read and Long-Read Sequencing Methods on Colorectal Cancer Samples.

Nikolett Szakállas1,2, Alexandra Kalmár2, Kristóf Róbert Rada2

  • 1Department of Biological Physics, Faculty of Science, Eötvös Loránd University, 1117 Budapest, Hungary.

International Journal of Molecular Sciences
|September 27, 2025
PubMed
Summary
This summary is machine-generated.

This study compares short-read Illumina and long-read Nanopore sequencing for colorectal cancer (CRC) genomics. Nanopore sequencing excels at detecting structural variants and preserving epigenetic signals, complementing Illumina

Keywords:
colorectal cancercomparative studylong-read sequencingmethodologynext-generation sequencingshort-read sequencing

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Area of Science:

  • Genomic sequencing technologies
  • Cancer genomics
  • Molecular diagnostics

Background:

  • Colorectal cancer (CRC) exhibits significant tumor heterogeneity and therapy resistance due to complex somatic mutations and structural variants.
  • Accurate detection of these genomic alterations is crucial for understanding CRC progression and developing effective treatments.

Purpose of the Study:

  • To comparatively analyze the performance of short-read Illumina and long-read Nanopore sequencing platforms for colorectal cancer genomics.
  • To evaluate base-level metrics and variant calling performance, including structural variants (SVs) and clinically relevant mutations.
  • To assess the utility of Nanopore sequencing for integrated genomic and epigenomic profiling in CRC.

Main Methods:

  • Comparative analysis of Illumina short-read and Nanopore long-read sequencing data from diverse colorectal cancer samples.
  • Evaluation of general sequencing metrics (nucleotide ratios, match rates, coverage) and variant calling performance (VAF, mutation detection).
  • Focus on clinically relevant genes (KRAS, BRAF, TP53, APC, PIK3CA) and structural variant analysis; assessment of PCR-free protocols for epigenomic profiling.

Main Results:

  • Nanopore sequencing demonstrated superior capability in resolving large and complex structural variants in CRC.
  • Both platforms showed distinct detection profiles for specific mutations; Nanopore sequencing preserved methylation signals with PCR-free protocols.
  • High-coverage Nanopore runs and exonic reference files facilitated direct comparison and accurate variant assessment.

Conclusions:

  • Short-read and long-read sequencing platforms offer complementary strengths for high-resolution cancer genomics.
  • Nanopore sequencing is valuable for integrated genomic and epigenomic profiling in colorectal cancer research.
  • Rigorous benchmarking, coverage normalization, and attention to epigenetic fidelity are essential for accurate variant discovery in cancer genomics.