Cell-Free DNA Blood Collection Tubes Crosslinking Cellular DNA Impeding Nanopore Long-Read Sequencing

Area of Science:

• Genomics
• Molecular Biology
• Cancer Research

Background:

• Long-read sequencing (DNA and RNA) is crucial for genome assembly, variant calling, and gene isoform identification.
• Long-read nanopore DNA sequencing is increasingly utilized in cancer research for structural variant calling.
• High molecular weight (HMW) DNA extraction from tumor and matched normal samples (blood/buffy coat) is standard for distinguishing somatic from germline mutations.

Purpose of the Study:

• To investigate the underperformance of buffy coat DNA compared to tumor DNA in long-read nanopore sequencing.
• To determine if formaldehyde release in Streck tubes causes DNA crosslinking, impacting sequencing data.
• To optimize DNA extraction protocols for buffy coat samples used in nanopore sequencing.

Main Methods:

• Comparison of DNA extraction yields and sequencing performance between tumor tissue and buffy coat samples.
• Analysis of DNA extracted from buffy coat cells collected in Streck tubes versus ethylenediaminetetraacetic acid (EDTA) tubes.
• Implementation and evaluation of a decrosslinking step during DNA extraction from Streck tube buffy coat samples.

Main Results:

• Buffy coat DNA consistently underperformed compared to tumor DNA in long-read nanopore sequencing.
• This underperformance was specific to buffy coat DNA collected in Streck tubes, not EDTA tubes.
• A decrosslinking step during DNA extraction significantly improved data yield and fragment length without compromising data quality.

Conclusions:

• Formaldehyde release in Streck tubes likely causes DNA crosslinking, hindering nanopore sequencing performance.
• A tailored DNA extraction protocol including a decrosslinking step is recommended for Streck-derived buffy coat samples.
• Optimized protocols enhance the utility of buffy coat samples for long-read nanopore sequencing in cancer research.