Advanced Research Department, Illumina, San Diego, California, USA.
Technology Development Department, Illumina, Little Chesterford, Essex, UK.
Nat Biotechnol. 2017 Sep;35(9):852-857. doi: 10.1038/nbt.3897. Epub 2017 Jun 26.
Haplotype-resolved genome sequencing promises to unlock a wealth of information in population and medical genetics. However, for the vast majority of genomes sequenced to date, haplotypes have not been determined because of cumbersome haplotyping workflows that require fractions of the genome to be sequenced in a large number of compartments. Here we demonstrate barcode partitioning of long DNA molecules in a single compartment using "on-bead" barcoded tagmentation. The key to the method that we call "contiguity preserving transposition" sequencing on beads (CPTv2-seq) is transposon-mediated transfer of homogenous populations of barcodes from beads to individual long DNA molecules that get fragmented at the same time (tagmentation). These are then processed to sequencing libraries wherein all sequencing reads originating from each long DNA molecule share a common barcode. Single-tube, bulk processing of long DNA molecules with ∼150,000 different barcoded bead types provides a barcode-linked read structure that reveals long-range molecular contiguity. This technology provides a simple, rapid, plate-scalable and automatable route to accurate, haplotype-resolved sequencing, and phasing of structural variants of the genome.
单倍型解析基因组测序有望在群体和医学遗传学中解锁大量信息。然而，由于繁琐的单倍型分析工作流程需要将基因组的一部分在大量隔室中进行测序，因此迄今为止对绝大多数测序的基因组尚未确定单倍型。在这里，我们展示了使用“珠上”带条码的标签酶切在单个隔室中对长 DNA 分子进行条码分区。我们称之为“珠上连续转座酶切测序”（CPTv2-seq）的方法的关键是转座酶介导的从珠上同质条码种群转移到同时进行片段化的单个长 DNA 分子（标签酶切）。然后将这些片段处理成测序文库，其中来自每个长 DNA 分子的所有测序读数都共享一个共同的条码。使用约 150,000 种不同带条码的珠类型进行长 DNA 分子的单管、批量处理提供了一种条码链接的读结构，揭示了长程分子连续性。这项技术为准确的、单倍型解析测序以及基因组结构变异的相位提供了一种简单、快速、板载可扩展和自动化的途径。
