Chamberlin John T, Gillen Austin E, Quinlan Aaron R
Department of Biomedical Informatics, University of Utah School of Medicine, 421 Wakara Way #140, Salt Lake City, UT 84112, USA.
RNA Bioscience Initiative, University of Colorado School of Medicine, 12801 E 17th Ave, Aurora, CO 80045, USA.
NAR Genom Bioinform. 2024 Dec 18;6(4):lqae175. doi: 10.1093/nargab/lqae175. eCollection 2024 Dec.
Prevailing poly(dT)-primed 3' single-cell RNA-seq protocols generate barcoded cDNA fragments containing the reverse transcriptase priming site or in principle the polyadenylation site. Direct sequencing across this site was historically difficult because of DNA sequencing errors induced by the homopolymeric primer at the 'barcode' end. Here, we evaluate the capability of 'avidity base chemistry' DNA sequencing from Element Biosciences to sequence through the primer and enable accurate paired-end read alignment and precise quantification of polyadenylation sites. We find that the Element Aviti instrument sequences through the thymine homopolymer into the subsequent cDNA sequence without detectable loss of accuracy. The additional sequence enables direct and independent assignment of reads to polyadenylation sites, which bypasses the complexities and limitations of conventional approaches but does not consistently improve read mapping rates compared to single-end alignment. We also characterize low-level artifacts and demonstrate necessary adjustments to adapter trimming and sequence alignment regardless of platform, particularly in the context of extended read lengths. Our analyses confirm that Element avidity sequencing is an effective alternative to Illumina sequencing for standard single-cell RNA-seq, particularly for polyadenylation site measurement but do not rule out the potential for similar performance from other emerging platforms.
目前流行的以聚（dT）为引物的3'单细胞RNA测序方案会生成包含逆转录酶引物位点或原则上包含聚腺苷酸化位点的带条形码的cDNA片段。由于“条形码”末端的同聚物引物会导致DNA测序错误，因此过去很难直接对该位点进行测序。在这里，我们评估了Element Biosciences公司的“亲和力碱基化学”DNA测序技术对引物进行测序的能力，该技术能够实现准确的双端读段比对以及对聚腺苷酸化位点进行精确量化。我们发现，Element Aviti仪器能够穿过胸腺嘧啶同聚物对后续的cDNA序列进行测序，且准确性没有明显损失。额外的序列能够将读段直接且独立地定位到聚腺苷酸化位点，这绕过了传统方法的复杂性和局限性，但与单端比对相比，读段映射率并没有持续提高。我们还对低水平的伪影进行了表征，并证明了无论使用何种平台，尤其是在读长延长的情况下，对接头修剪和序列比对进行必要调整的必要性。我们的分析证实，对于标准的单细胞RNA测序，尤其是聚腺苷酸化位点的测量，Element亲和力测序是Illumina测序的有效替代方法，但不排除其他新兴平台具有类似性能的可能性。
