Deng Dori Z Q, Verhage Jack, Neudorf Celine, Corbett-Detig Russell, Mekonen Honey, Castaldi Peter J, Vollmers Christopher
Department of Molecular, Cellular, and Developmental Biology, University of California Santa Cruz, Santa Cruz, CA 95064, USA.
Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, CA 95064, USA.
PNAS Nexus. 2024 Aug 21;3(9):pgae336. doi: 10.1093/pnasnexus/pgae336. eCollection 2024 Sep.
The sequencing of PCR amplicons is a core application of high-throughput sequencing technology. Using unique molecular identifiers (UMIs), individual amplified molecules can be sequenced to very high accuracy on an Illumina sequencer. However, Illumina sequencers have limited read length and are therefore restricted to sequencing amplicons shorter than 600 bp unless using inefficient synthetic long-read approaches. Native long-read sequencers from Pacific Biosciences and Oxford Nanopore Technologies can, using consensus read approaches, match or exceed Illumina quality while achieving much longer read lengths. Using a circularization-based concatemeric consensus sequencing approach (R2C2) paired with UMIs (R2C2 + UMI), we show that we can sequence an ∼550-nt antibody heavy chain (Immunoglobulin heavy chain - IGH) and an ∼1,500-nt 16S amplicons at accuracies up to and exceeding Q50 (<1 error in 100,000 sequenced bases), which exceeds accuracies of UMI-supported Illumina-paired sequencing as well as synthetic long-read approaches.
PCR扩增子测序是高通量测序技术的核心应用。使用独特分子标识符（UMIs），单个扩增分子可在Illumina测序仪上以非常高的准确性进行测序。然而，Illumina测序仪的读长有限，因此除非使用效率低下的合成长读长方法，否则仅限于对短于600 bp的扩增子进行测序。太平洋生物科学公司和牛津纳米孔技术公司的原生长读长测序仪可以使用一致性读长方法，在实现更长读长的同时达到或超过Illumina的质量。使用基于环化的串联一致性测序方法（R2C2）与UMIs（R2C2 + UMI）相结合，我们表明我们可以对一个约550 nt的抗体重链（免疫球蛋白重链 - IGH）和一个约1500 nt的16S扩增子进行测序，准确性高达并超过Q50（在100,000个测序碱基中误差小于1个），这超过了UMI支持的Illumina配对测序以及合成长读长方法的准确性。
