A ligation-based single-stranded library preparation method to analyze cell-free DNA and synthetic oligos

BMC Genomics

Kelly M. Harkins¹  Jessica M. Morgan¹  Colin Naughton¹  Christopher J. Troll¹  Varsha Rao¹  Richard E. Green²  Charles Cole²  Beth Shapiro³  Joshua Kapp³ 

[1] Claret Bioscience LLC;Department of Biomolecular Engineering, University of California Santa Cruz;Department of Ecology and Evolutionary Biology, University of California;

关键词: SRSLY;    Single-stranded library;    Next-generation sequencing;    Cell-free DNA;    Oligos;    Nucleosome positioning;   

DOI  :  10.1186/s12864-019-6355-0

来源: DOAJ

【 摘 要 】

Abstract Background Cell-free DNA (cfDNA), present in circulating blood plasma, contains information about prenatal health, organ transplant reception, and cancer presence and progression. Originally developed for the genomic analysis of highly degraded ancient DNA, single-stranded DNA (ssDNA) library preparation methods are gaining popularity in the field of cfDNA analysis due to their efficiency and ability to convert short, fragmented DNA into sequencing libraries without altering DNA ends. However, current ssDNA methods are costly and time-consuming. Results Here we present an efficient ligation-based single-stranded library preparation method that is engineered to produce complex libraries in under 2.5 h from as little as 1 nanogram of input DNA without alteration to the native ends of template molecules. Our method, called Single Reaction Single-stranded LibrarY or SRSLY, ligates uniquely designed Next-Generation Sequencing (NGS) adapters in a one-step combined phosphorylation/ligation reaction that foregoes end-polishing. Using synthetic DNA oligos and cfDNA, we demonstrate the efficiency and utility of this approach and compare with existing double-stranded and single-stranded approaches for library generation. Finally, we demonstrate that cfDNA NGS data generated from SRSLY can be used to analyze DNA fragmentation patterns to deduce nucleosome positioning and transcription factor binding. Conclusions SRSLY is a versatile tool for converting short and fragmented DNA molecules, like cfDNA fragments, into sequencing libraries while retaining native lengths and ends.

【 授权许可】

Unknown