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, California, USA.
Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, California, USA.
bioRxiv. 2023 Aug 21:2023.08.19.553937. doi: 10.1101/2023.08.19.553937.
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 600bp 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 ~550nt antibody heavy-chain (IGH) and ~1500nt 16S amplicons at accuracies up to and exceeding Q50 (<1 error in 100,0000 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），单个扩增分子可以在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600bp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，使用一致性读长方法，可以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）并结合UMIs（R2C2+UMI），我们表明我们可以对约550nt的抗体重链（IGH）和约1500nt的16S扩增子进行测序，准确率高达并超过Q50（每1000000个测序碱基中错误少于1个），这超过了UMI支持的Illumina双端测序以及合成长读长方法的准确率。
