Jaksik Roman, Drobna-Śledzińska Monika, Dawidowska Małgorzata
Department of Systems Biology and Engineering, Silesian University of Technology, Gliwice, Poland; Biotechnology Centre, Silesian University of Technology, Gliwice, Poland.
Institute of Human Genetics Polish Academy of Sciences, Strzeszyńska 32, 60-479 Poznań, Poland.
Genomics. 2021 Nov;113(6):4149-4162. doi: 10.1016/j.ygeno.2021.10.018. Epub 2021 Nov 3.
With long reads and high coverage, RNA-seq enables comprehensive transcriptome analysis of cancer cells, provided that optimal length of libraries (and their inserts) is assured, to avoid overlap of paired reads and consequent loss of sequencing data. We assessed TruSeq Stranded library preparation protocols (poly(A) enrichment-PA and rRNA depletion-RD) for the thoroughness of transcriptome analysis of a heterogeneous cancer, acute lymphoblastic leukemia. We applied 2x150PE sequencing, >150 M reads/sample on Illumina NovaSeq6000. We show that PA outperforms RD for the analysis of gene expression and structural aberrations. RD is more suitable for detection of various classes of RNAs, mutations or polymorphisms. We demonstrate that reduced RNA fragmentation time (generating longer inserts) positively affects detection of structural RNA changes, without introducing bias into gene expression analysis. We recommend this modification for all RNA-seq studies utilizing reads longer than 75 nt, aimed to go beyond gene expression analysis and to detect also structural changes.
凭借长读长和高覆盖率，RNA测序能够对癌细胞进行全面的转录组分析，前提是确保文库（及其插入片段）的最佳长度，以避免双端读段重叠并导致测序数据丢失。我们评估了TruSeq链特异性文库制备方案（聚腺苷酸富集法-PA和核糖体RNA去除法-RD）用于异质性癌症——急性淋巴细胞白血病转录组分析的彻底性。我们采用2×150PE测序，在Illumina NovaSeq6000上每个样本测序读数>1.5亿。我们发现，在基因表达和结构畸变分析方面，PA优于RD。RD更适合检测各类RNA、突变或多态性。我们证明，缩短RNA片段化时间（产生更长的插入片段）对结构RNA变化的检测有积极影响，且不会在基因表达分析中引入偏差。对于所有使用长度超过75 nt读长的RNA测序研究，若旨在超越基因表达分析并检测结构变化，我们推荐这种改进方法。
