Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis

Cleary, Michele A.; Kilian, Kristopher A.; Wang, Yanqun; Bradshaw, Jeffrey D.; Cavet, Guy; Ge, Wei; Kulkarni, Amit; Paddison, Patrick J.; Chang, Kenneth; Sheth, Nihar U.; LeProust, Eric; Coffey, Ernest M.; Burchard, Julja; McCombie, W. Richard; Linsley, Peter S.; Hannon, Gregory J.

doi:10.1038/nmeth724

Cited by 100 publications

(87 citation statements)

References 33 publications

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“…In the shRNA scenario, 40,320 simulated bacterial clones carried one of 12,000-17,000 possible inserts, a number consistent with array-based oligonucleotide library synthesis (Cleary et al 2004). Based on prior experience, we estimated d 0 to be no more than 40-50, even in the presence of biased cloning.…”

Section: Dna Sudokumentioning

confidence: 89%

“…Our approach relies on highly parallel oligonucleotide synthesis (Cleary et al 2004) to generate mixtures of approximately 22,000-55,000 different species that are adapted and ligated into expression vectors. Previously, individual clones from this mixed library were picked, arrayed, and conventionally sequenced both to determine their identity and to verify their accuracy.…”

Section: Conceptual Framework For the Strategymentioning

confidence: 99%

See 1 more Smart Citation

DNA Sudoku—harnessing high-throughput sequencing for multiplexed specimen analysis

Erlich¹,

Chang²,

Gordon³

et al. 2009

Genome Res.

Self Cite

147

187

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Next-generation sequencers have sufficient power to analyze simultaneously DNAs from many different specimens, a practice known as multiplexing. Such schemes rely on the ability to associate each sequence read with the specimen from which it was derived. The current practice of appending molecular barcodes prior to pooling is practical for parallel analysis of up to many dozen samples. Here, we report a strategy that permits simultaneous analysis of tens of thousands of specimens. Our approach relies on the use of combinatorial pooling strategies in which pools rather than individual specimens are assigned barcodes. Thus, the identity of each specimen is encoded within the pooling pattern rather than by its association with a particular sequence tag. Decoding the pattern allows the sequence of an original specimen to be inferred with high confidence. We verified the ability of our encoding and decoding strategies to accurately report the sequence of individual samples within a large number of mixed specimens in two ways. First, we simulated data both from a clone library and from a human population in which a sequence variant associated with cystic fibrosis was present. Second, we actually pooled, sequenced, and decoded identities within two sets of 40,000 bacterial clones comprising approximately 20,000 different artificial microRNAs targeting Arabidopsis or human genes. We achieved greater than 97% accuracy in these trials. The strategies reported here can be applied to a wide variety of biological problems, including the determination of genotypic variation within large populations of individuals.[Supplemental material is available online at www.genome.org.]There are many biological applications wherein the sequence of a small region must be determined from many independent specimens and wherein the link between the specimen and the sequence must be preserved. Examples might include studies of variation within specific regions from a large population, looking for correlations between variants and phenotype in areas that might have been narrowed by prior linkage analysis, or even medical diagnostics. We were faced with the practical matter of determining clone identity and quality in a very large collection of arrayed clones derived from a pooled oligonucleotide mixture. Use of next-generation sequencing technologies (NGST) for interrogating sequence for any of these tasks is hampered if libraries must be constructed independently from each sample or if different barcodes must be applied to each specimen prior to sequencing, particularly if specimen numbers run into many thousands.To circumvent such limitations, we have developed a novel multiplexing framework in which the identity of individual specimens is not encoded directly by an appended barcode sequence. Instead, specimen identities are encoded by positional information created by an optimized series of pooling schema. In this manner, more than 100,000 different samples can be analyzed using only a few hundred barcodes (e.g., 384). Results Conceptual fr...

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Section: Dna Sudokumentioning

confidence: 89%

Section: Conceptual Framework For the Strategymentioning

confidence: 99%

DNA Sudoku—harnessing high-throughput sequencing for multiplexed specimen analysis

Erlich¹,

Chang²,

Gordon³

et al. 2009

Genome Res.

Self Cite

147

187

View full text Add to dashboard Cite

show abstract

“…Alternatively, ink-jet printing-based technologies developed by Agilent and semiconductor-based chip technologies by CombiMatrix (CustomArray) allow for the use of standard phosphoramidites and reagents without the need for expensive micromirror controllers or photomasking. In particular, these two synthesis technologies currently provide the best combination of cost, oligonucleotide synthesis length, and accuracy, which has led to their use in several recent gene synthesis applications (Cleary et al 2004;Warner et al 2010;Patwardhan et al 2012). Using microarraybased synthesizers, oligonucleotides are typically synthesized at femtomolar synthesis scales, that is, typically two to four orders of magnitude lower than that used in column-based synthesis.…”

Section: Microarray-based Oligonucleotide Synthesismentioning

confidence: 99%

Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology

Hughes

Ellington

2017

Cold Spring Harb Perspect Biol

322

235

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The chemical synthesis of DNA oligonucleotides and their assembly into synthons, genes, circuits, and even entire genomes by gene synthesis methods has become an enabling technology for modern molecular biology and enables the design, build, test, learn, and repeat cycle underpinning innovations in synthetic biology. In this perspective, we briefly review the techniques and technologies that enable the synthesis of DNA oligonucleotides and their assembly into larger DNA constructs with a focus on recent advancements that have sought to reduce synthesis cost and increase sequence fidelity. The development of lower-cost methods to produce high-quality synthetic DNA will allow for the exploration of larger biological hypotheses by lowering the cost of use and help to close the DNA read -write cost gap.DNA neither cares nor knows. DNA just is. And we dance to its music.-Richard Dawkins River Out of Eden: A Darwinian Life

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“…To ensure that individual shRNAs were cloned together with their specific Sensor, we applied large-scale onchip oligonucleotide synthesis (Cleary et al, 2004) to produce $20,000 185-mers, each harboring an shRNA and its target sequence separated by cloning sites, and used them to assemble the Sensor library in a pooled two-step procedure (Figure 2A). Serving as internal controls, all 17 previously characterized shRNAs were included at 15-fold representation to ensure their presence in the final pool.…”

Section: Generation Of a High-complexity Sensor Librarymentioning

confidence: 99%

RNAi as a Routine Route Toward Breast Cancer Therapy

Hannon¹

2014

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Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. REPORT DATE (DD-MM-YYYY)May 2014 (From -To) 1Sep2008-28Feb2014 REPORT TYPE Final DATES COVERED TITLE AND SUBTITLERNAi as a Routine Route Toward Breast Cancer Therapy 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-08-1-0572 5c. PROGRAM ELEMENT NUMBER AUTHOR(S)Gregory J. Hannon, PhD. 5d. PROJECT NUMBERemail: hannon@cshl.edu 5e. TASK NUMBER 5f. WORK UNIT NUMBER PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBERCold Spring Harbor Laboratory One Bungtown Road Cold Spring Harbor, NY 11724 SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR'S ACRONYM(S)US Army Medical Research and Materiel Command Fort Detrick, MA 21702 SPONSOR/MONITOR'S REPORT NUMBER(S) DISTRIBUTION / AVAILABILITY STATEMENTApproved for public release; distribution unlimited SUPPLEMENTARY NOTES ABSTRACTDuring the first year of this innovator award, we made significant progress toward two of our aims. We constructed a third generation RNAi library and made that available to the breast cancer community. This resource has nearly 75,000 independent, sequence verified clones targeting ~18,000 human genes. A similar library for the mouse genome is nearing completion. We also scaled up our shRNA screening platform in preparation for lethality surveys of all suitable and available BC cell lines, including matched pairs of lines that have acquired resistance to herceptin in vitro. Relevant to our second aim, we have profiled microRNA from each of the identifiable epithelial cell types in the mouse mammary gland and are undertaking similar efforts in human. The goal is to develop microRNA sensor strategies that will permit visualization of each cell type in vivo and enable their isolation and manipulation in vitro. Finally, we showed that two microRNAs, let-7 and miR-93, can deplete tumor initiating cells from a number of basal breast cancer cell lines. SUBJECT TERMS INTRODUCTIONThe goal of this innovator award is to continue to develop and apply RNAi-based screening methods to discover new routes toward breast cancer therapy. The project has three sets of goals. First is to integrate genomic and geneti...

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Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis

Cited by 100 publications

References 33 publications

DNA Sudoku—harnessing high-throughput sequencing for multiplexed specimen analysis

DNA Sudoku—harnessing high-throughput sequencing for multiplexed specimen analysis

Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology

RNAi as a Routine Route Toward Breast Cancer Therapy

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