Tagmentation method for preparing DNA for NGS sequencing from as little as 20pg input DNA amount, as developed by the Systems Biology Laboratory UK.

New sequencing technologies can address diverse biomedical questions but are limited by a minimum required DNA input of typically 1 microgram. We describe how sequencing libraries can be reproducibly created from 20pg input DNA using a modified transpososome-mediated fragmentation technique. Resulting libraries incorporate in-line barcoding which facilitates sample multiplexes that can be sequenced using Illumina platforms with the manufacturer's sequencing primer. We demonstrate this technique by providing deep coverage sequence of the E. coli K-12 genome that shows equivalent target coverage to a 1mg input library prepared using standard Illumina methods. Reducing template quantity does, however, increase the proportion of duplicate reads and enriches coverage in low GC regions. This finding was confirmed with low coverage re-sequencing of mouse from 20pg gDNA input, equivalent to ~7x haploid genomes, where ~0.4x coverage of mapped fragments were recovered. Application of this new method now allows genomic studies from low mass samples and routine preparation of sequencing libraries from enrichment procedures.

新兴测序技术可应对多样化的生物医学研究问题，但普遍受限于最低需投入约1微克的DNA样本。本研究详述了如何利用修饰型转座体介导片段化技术，从仅20皮克的起始DNA中可重复构建测序文库。所得测序文库集成内嵌式条形码技术，可实现样本多重测序，且可使用Illumina测序平台及其官方测序引物完成测序。本研究通过对大肠杆菌K-12（E. coli K-12）基因组开展深度覆盖测序，验证了该技术的有效性：其目标区域覆盖度与采用标准Illumina方法构建的1毫克起始DNA文库相当。不过，降低起始模板量会提升重复读段的占比，并会使低GC含量区域的测序覆盖度出现富集。该结论通过小鼠基因组DNA（gDNA）的低覆盖度重测序实验得到验证：以20皮克的小鼠基因组DNA为起始样本（约相当于7倍单倍体基因组总量），最终回收的比对片段覆盖度约为0.4倍。该新技术的应用，使得从微量样本中开展基因组研究成为可能，同时也可实现富集实验产物的测序文库常规构建。