Abstract
BackgroundCell-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.ResultsHere 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.ConclusionsSRSLY is a versatile tool for converting short and fragmented DNA molecules, like cfDNA fragments, into sequencing libraries while retaining native lengths and ends.
Highlights
Cell-free DNA, present in circulating blood plasma, contains information about prenatal health, organ transplant reception, and cancer presence and progression
Developed for the genomic analysis of highly degraded ancient DNA [7, 8], single-stranded DNA (ssDNA) library preparation methods have been adopted for other fragmented sample types such as such as cell-free DNA and DNA purified from Formalin Fixed Paraffin Embedded (FFPE) sections, due to their efficiency in converting a high fraction of input DNA fragments into sequencing library molecules and their ability to capture small DNA fragments
We present standard sequencing metrics produced by SRSLY libraries made with Cell-free DNA (cfDNA) from healthy human donors and compare our results to those of commercially available library preparation methods
Summary
Cell-free DNA (cfDNA), present in circulating blood plasma, contains information about prenatal health, organ transplant reception, and cancer presence and progression. 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. Conventional methods for Next-Generation Sequencing (NGS) library preparation convert only double-stranded DNA (dsDNA) into library-ready molecules. Developed for the genomic analysis of highly degraded ancient DNA [7, 8], ssDNA library preparation methods have been adopted for other fragmented sample types such as such as cell-free DNA (cfDNA) and DNA purified from Formalin Fixed Paraffin Embedded (FFPE) sections, due to their efficiency in converting a high fraction of input DNA fragments into sequencing library molecules and their ability to capture small DNA fragments. When mapped to a reference genome, these data reveal the exact genomic location of the input fragments; an important feature for cfDNA researchers studying biological fragmentation patterns
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