Chrysanthou Stephanie, Karmacharya Trishala, Li Juan, Lu Cuijie, Cobbs Cassidy C, Mohibullah Neeman
Integrated Genomics Operation Memorial Sloan Kettering Cancer Center.
J Biomol Tech. 2025 Mar 19;36(1). doi: 10.7171/3fc1f5fe.ad7e097e. eCollection 2025 Apr 30.
Long-read DNA and RNA sequencing facilitate genome assembly, haplotyping, complex variant calling, and gene isoform identification. Structural variant calling is integral to the molecular characterization of tumors; thus, long-read nanopore DNA sequencing technology is becoming routinely used in cancer research. As a standard practice, high molecular weight (HMW) DNA is extracted from both tumor and matched normal samples from blood or buffy coat to redact germline variants from somatic mutations. However, we found that buffy coat DNA consistently underperformed compared to DNA extracted from tumor tissue. Furthermore, this observation was unique to DNA extracted from buffy coat cells collected in Streck, but not ethylenediaminetetraacetic acid (EDTA), tubes. We therefore investigated whether the released formaldehyde in Streck tubes resulted in DNA crosslinking, which would explain the low data throughput. Indeed, a decrosslinking step during Streck DNA extraction significantly improved data yield and fragment length without compromising data quality. We therefore recommend a tailored DNA extraction protocol of Streck- derived buffy coat samples for nanopore sequencing.
长读长DNA和RNA测序有助于基因组组装、单倍型分型、复杂变异检测和基因异构体鉴定。结构变异检测是肿瘤分子特征分析不可或缺的一部分；因此，长读长纳米孔DNA测序技术正逐渐在癌症研究中得到常规应用。作为标准做法，从肿瘤以及血液或血沉棕黄层的匹配正常样本中提取高分子量（HMW）DNA，以从体细胞突变中剔除种系变异。然而，我们发现，与从肿瘤组织中提取的DNA相比，血沉棕黄层DNA的表现始终较差。此外，这一观察结果对于从Streck管中收集的血沉棕黄层细胞提取的DNA是独特的，但对于从乙二胺四乙酸（EDTA）管中收集的细胞提取的DNA则并非如此。因此，我们研究了Streck管中释放的甲醛是否会导致DNA交联，这可以解释数据通量较低的原因。事实上，在Streck DNA提取过程中的去交联步骤显著提高了数据产量和片段长度，同时不影响数据质量。因此，我们建议针对纳米孔测序采用一种专门的从Streck来源的血沉棕黄层样本中提取DNA的方案。
