Abstract
BackgroundTransposome-based technologies have enabled the streamlined production of sequencer-ready DNA libraries; however, current methods are highly sensitive to the amount and quality of input nucleic acid.ResultsWe describe a new library preparation technology (Nextera DNA Flex) that utilizes a known concentration of transposomes conjugated directly to beads to bind a fixed amount of DNA, and enables direct input of blood and saliva using an integrated extraction protocol. We further report results from libraries generated outside the standard parameters of the workflow, highlighting novel applications for Nextera DNA Flex, including human genome builds and variant calling from below 1 ng DNA input, customization of insert size, and preparation of libraries from short fragments and severely degraded FFPE samples. Using this bead-linked library preparation method, library yield saturation was observed at an input amount of 100 ng. Preparation of libraries from a range of species with varying GC levels demonstrated uniform coverage of small genomes. For large and complex genomes, coverage across the genome, including difficult regions, was improved compared with other library preparation methods. Libraries were successfully generated from amplicons of varying sizes (from 50 bp to 11 kb), however, a decrease in efficiency was observed for amplicons smaller than 250 bp. This library preparation method was also compatible with poor-quality DNA samples, with sequenceable libraries prepared from formalin-fixed paraffin-embedded samples with varying levels of degradation.ConclusionsIn contrast to solution-based library preparation, this bead-based technology produces a normalized, sequencing-ready library for a wide range of DNA input types and amounts, largely obviating the need for DNA quantitation. The robustness of this bead-based library preparation kit and flexibility of input DNA facilitates application across a wide range of fields.
Highlights
Transposome-based technologies have enabled the streamlined production of sequencer-ready DNA libraries; current methods are highly sensitive to the amount and quality of input nucleic acid
In binding a fixed amount of DNA, the immobilized transposomes should fragment the DNA to a set size distribution, leading to library yield normalization at a saturating DNA input amount
We determined that the beads become saturated at an input amount of around 100 ng, with normalized yields of around 330 ng observed for inputs of 100 ng to 1 μg (Fig. 1a); for DNA inputs less than 100 ng, the library yield was directly correlated to the input amount
Summary
Transposome-based technologies have enabled the streamlined production of sequencer-ready DNA libraries; current methods are highly sensitive to the amount and quality of input nucleic acid. Library preparation is an important first step for all next-generation sequencing (NGS) applications and generally employs several common steps. In vitro transposition has been used to Bruinsma et al BMC Genomics (2018) 19:722 process that can have downstream effects on sequencing coverage. We describe a new library preparation technology, which employs magnetic-bead linked transposomes (BLT). Using a known quantity of transposomes conjugated directly to beads fixes the amount of DNA (for inputs of at least 100 ng), allowing libraries with consistent fragment sizes and yields to be generated in a few hours. We have optimized the methodology to maximize library diversity, which results in improved coverage uniformity across the genome. We illustrate the broad applicability of this method, from amplicons and small genomes to large or complex genomes
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