A method for high‐throughput production of sequence‐verified DNA libraries and strain collections
Abstract The low costs of array‐synthesized oligonucleotide libraries are empowering rapid advances in quantitative and synthetic biology. However, high synthesis error rates, uneven representation, and lack of access to individual oligonucleotides limit the true potential of these libraries. We hav...
| Main Authors: | , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Springer Nature
2017-02-01
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| Series: | Molecular Systems Biology |
| Subjects: | |
| Online Access: | https://doi.org/10.15252/msb.20167233 |
| _version_ | 1797279829900394496 |
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| author | Justin D Smith Ulrich Schlecht Weihong Xu Sundari Suresh Joe Horecka Michael J Proctor Raeka S Aiyar Richard A O Bennett Angela Chu Yong Fuga Li Kevin Roy Ronald W Davis Lars M Steinmetz Richard W Hyman Sasha F Levy Robert P St.Onge |
| author_facet | Justin D Smith Ulrich Schlecht Weihong Xu Sundari Suresh Joe Horecka Michael J Proctor Raeka S Aiyar Richard A O Bennett Angela Chu Yong Fuga Li Kevin Roy Ronald W Davis Lars M Steinmetz Richard W Hyman Sasha F Levy Robert P St.Onge |
| author_sort | Justin D Smith |
| collection | DOAJ |
| description | Abstract The low costs of array‐synthesized oligonucleotide libraries are empowering rapid advances in quantitative and synthetic biology. However, high synthesis error rates, uneven representation, and lack of access to individual oligonucleotides limit the true potential of these libraries. We have developed a cost‐effective method called Recombinase Directed Indexing (REDI), which involves integration of a complex library into yeast, site‐specific recombination to index library DNA, and next‐generation sequencing to identify desired clones. We used REDI to generate a library of ~3,300 DNA probes that exhibited > 96% purity and remarkable uniformity (> 95% of probes within twofold of the median abundance). Additionally, we created a collection of ~9,000 individually accessible CRISPR interference yeast strains for > 99% of genes required for either fermentative or respiratory growth, demonstrating the utility of REDI for rapid and cost‐effective creation of strain collections from oligonucleotide pools. Our approach is adaptable to any complex DNA library, and fundamentally changes how these libraries can be parsed, maintained, propagated, and characterized. |
| first_indexed | 2024-03-07T16:31:39Z |
| format | Article |
| id | doaj.art-7ab84b1308cd4c04b4426d5c0715d2a2 |
| institution | Directory Open Access Journal |
| issn | 1744-4292 |
| language | English |
| last_indexed | 2024-03-07T16:31:39Z |
| publishDate | 2017-02-01 |
| publisher | Springer Nature |
| record_format | Article |
| series | Molecular Systems Biology |
| spelling | doaj.art-7ab84b1308cd4c04b4426d5c0715d2a22024-03-03T10:19:58ZengSpringer NatureMolecular Systems Biology1744-42922017-02-01132n/an/a10.15252/msb.20167233A method for high‐throughput production of sequence‐verified DNA libraries and strain collectionsJustin D Smith0Ulrich Schlecht1Weihong Xu2Sundari Suresh3Joe Horecka4Michael J Proctor5Raeka S Aiyar6Richard A O Bennett7Angela Chu8Yong Fuga Li9Kevin Roy10Ronald W Davis11Lars M Steinmetz12Richard W Hyman13Sasha F Levy14Robert P St.Onge15Stanford Genome Technology Center Stanford University Palo Alto CA USAStanford Genome Technology Center Stanford University Palo Alto CA USAStanford Genome Technology Center Stanford University Palo Alto CA USAStanford Genome Technology Center Stanford University Palo Alto CA USAStanford Genome Technology Center Stanford University Palo Alto CA USAStanford Genome Technology Center Stanford University Palo Alto CA USAStanford Genome Technology Center Stanford University Palo Alto CA USALaufer Center for Physical and Quantitative Biology Stony Brook University Stony Brook NY USAStanford Genome Technology Center Stanford University Palo Alto CA USAStanford Genome Technology Center Stanford University Palo Alto CA USAStanford Genome Technology Center Stanford University Palo Alto CA USAStanford Genome Technology Center Stanford University Palo Alto CA USAStanford Genome Technology Center Stanford University Palo Alto CA USAStanford Genome Technology Center Stanford University Palo Alto CA USALaufer Center for Physical and Quantitative Biology Stony Brook University Stony Brook NY USAStanford Genome Technology Center Stanford University Palo Alto CA USAAbstract The low costs of array‐synthesized oligonucleotide libraries are empowering rapid advances in quantitative and synthetic biology. However, high synthesis error rates, uneven representation, and lack of access to individual oligonucleotides limit the true potential of these libraries. We have developed a cost‐effective method called Recombinase Directed Indexing (REDI), which involves integration of a complex library into yeast, site‐specific recombination to index library DNA, and next‐generation sequencing to identify desired clones. We used REDI to generate a library of ~3,300 DNA probes that exhibited > 96% purity and remarkable uniformity (> 95% of probes within twofold of the median abundance). Additionally, we created a collection of ~9,000 individually accessible CRISPR interference yeast strains for > 99% of genes required for either fermentative or respiratory growth, demonstrating the utility of REDI for rapid and cost‐effective creation of strain collections from oligonucleotide pools. Our approach is adaptable to any complex DNA library, and fundamentally changes how these libraries can be parsed, maintained, propagated, and characterized.https://doi.org/10.15252/msb.20167233arrayed strain collectionCRISPR interferenceDNA librariesoligonucleotide poolssynthetic biology |
| spellingShingle | Justin D Smith Ulrich Schlecht Weihong Xu Sundari Suresh Joe Horecka Michael J Proctor Raeka S Aiyar Richard A O Bennett Angela Chu Yong Fuga Li Kevin Roy Ronald W Davis Lars M Steinmetz Richard W Hyman Sasha F Levy Robert P St.Onge A method for high‐throughput production of sequence‐verified DNA libraries and strain collections Molecular Systems Biology arrayed strain collection CRISPR interference DNA libraries oligonucleotide pools synthetic biology |
| title | A method for high‐throughput production of sequence‐verified DNA libraries and strain collections |
| title_full | A method for high‐throughput production of sequence‐verified DNA libraries and strain collections |
| title_fullStr | A method for high‐throughput production of sequence‐verified DNA libraries and strain collections |
| title_full_unstemmed | A method for high‐throughput production of sequence‐verified DNA libraries and strain collections |
| title_short | A method for high‐throughput production of sequence‐verified DNA libraries and strain collections |
| title_sort | method for high throughput production of sequence verified dna libraries and strain collections |
| topic | arrayed strain collection CRISPR interference DNA libraries oligonucleotide pools synthetic biology |
| url | https://doi.org/10.15252/msb.20167233 |
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