Bead-linked transposomes enable a normalization-free workflow for NGS library preparation
Abstract Background Transposome-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. Results We describe a new library preparation technology (Nextera DNA Flex) that...
| Main Authors: | , , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2018-10-01
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| Series: | BMC Genomics |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12864-018-5096-9 |
| _version_ | 1819076154285359104 |
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| author | Stephen Bruinsma Joshua Burgess Daniel Schlingman Agata Czyz Natalie Morrell Catherine Ballenger Heather Meinholz Lee Brady Anupama Khanna Lindsay Freeberg Rosamond G Jackson Pascale Mathonet Susan C Verity Andrew F Slatter Rooz Golshani Haiying Grunenwald Gary P Schroth Niall A Gormley |
| author_facet | Stephen Bruinsma Joshua Burgess Daniel Schlingman Agata Czyz Natalie Morrell Catherine Ballenger Heather Meinholz Lee Brady Anupama Khanna Lindsay Freeberg Rosamond G Jackson Pascale Mathonet Susan C Verity Andrew F Slatter Rooz Golshani Haiying Grunenwald Gary P Schroth Niall A Gormley |
| author_sort | Stephen Bruinsma |
| collection | DOAJ |
| description | Abstract Background Transposome-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. Results We 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. Conclusions In 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. |
| first_indexed | 2024-12-21T18:36:47Z |
| format | Article |
| id | doaj.art-90493b047f81402d9c34969cb9219954 |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-12-21T18:36:47Z |
| publishDate | 2018-10-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-90493b047f81402d9c34969cb92199542022-12-21T18:54:07ZengBMCBMC Genomics1471-21642018-10-0119111610.1186/s12864-018-5096-9Bead-linked transposomes enable a normalization-free workflow for NGS library preparationStephen Bruinsma0Joshua Burgess1Daniel Schlingman2Agata Czyz3Natalie Morrell4Catherine Ballenger5Heather Meinholz6Lee Brady7Anupama Khanna8Lindsay Freeberg9Rosamond G Jackson10Pascale Mathonet11Susan C Verity12Andrew F Slatter13Rooz Golshani14Haiying Grunenwald15Gary P Schroth16Niall A Gormley17Illumina, Inc.Illumina, Inc.Illumina, Inc.Illumina, Inc.Illumina, Inc.Illumina, Inc.Illumina, Inc.Illumina, Inc.Illumina, Inc.Illumina, Inc.Illumina, Inc.Illumina, Inc.Illumina, Inc.Illumina, Inc.Illumina, Inc.Illumina, Inc.Illumina, Inc.Illumina, Inc.Abstract Background Transposome-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. Results We 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. Conclusions In 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.http://link.springer.com/article/10.1186/s12864-018-5096-9Next-generation sequencingLibrary preparationTransposome |
| spellingShingle | Stephen Bruinsma Joshua Burgess Daniel Schlingman Agata Czyz Natalie Morrell Catherine Ballenger Heather Meinholz Lee Brady Anupama Khanna Lindsay Freeberg Rosamond G Jackson Pascale Mathonet Susan C Verity Andrew F Slatter Rooz Golshani Haiying Grunenwald Gary P Schroth Niall A Gormley Bead-linked transposomes enable a normalization-free workflow for NGS library preparation BMC Genomics Next-generation sequencing Library preparation Transposome |
| title | Bead-linked transposomes enable a normalization-free workflow for NGS library preparation |
| title_full | Bead-linked transposomes enable a normalization-free workflow for NGS library preparation |
| title_fullStr | Bead-linked transposomes enable a normalization-free workflow for NGS library preparation |
| title_full_unstemmed | Bead-linked transposomes enable a normalization-free workflow for NGS library preparation |
| title_short | Bead-linked transposomes enable a normalization-free workflow for NGS library preparation |
| title_sort | bead linked transposomes enable a normalization free workflow for ngs library preparation |
| topic | Next-generation sequencing Library preparation Transposome |
| url | http://link.springer.com/article/10.1186/s12864-018-5096-9 |
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