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( A ) Distribution of protein lengths for all entries in Swiss-Prot. Most proteins are too large to encode on single oligos, represented as the shaded gray area. Oligos provide ~250 bp of coding sequence given a 300-bp oligo with PCR and cloning adaptors. The <t>synthesis</t> cost per gene is shown above the distribution using <t>Twist</t> <t>Bioscience</t> prices for <t>fragment</t> synthesis. Structures are from RCSB Molecule of the Month articles and use the following structure codes from left to right: 1GFL, 4EYL, 5XH3, 1NW9, 1W0E, 2D1S, 1ASZ, and 4OO8. USD, US dollar. ( B ) OMEGA design and assembly workflow. Users provide a list of codon-optimized sequences that OMEGA randomly sorts into N subassembly pools. For each subpool, OMEGA designs fragments that use high-fidelity GG sites and ensures that all pieces are within oligo size constraints. Subassembly pools are separately amplified and assembled into full-length gene products in parallel, after which assembly products are combined to recover the designed library. ( C ) Effect of sequence identity on assembly fidelity. Average predicted assembly fidelity for subpools containing sequences with 50, ~88, or 100% pairwise identity. Each condition was tested using assemblies of 4 or 12 fragments and subpools containing 50 or 70 GG sites. ( D ) Effect of GC content on assembly fidelity. Average predicted assembly fidelity for synthetic sequences with GC content ranging from 20 to 80%. Assemblies were tested for both 4- and 12-fragment constructs and across varied sequence identities. ( E ) Assembly fidelity of repetitive sequences. Average predicted assembly fidelity for DARPin sequences composed of five ankyrin repeats, using 70 GG sites per subpool and varying sequence identities. Mean fidelities across all subpools differed by less than 1%.
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( A ) Distribution of protein lengths for all entries in Swiss-Prot. Most proteins are too large to encode on single oligos, represented as the shaded gray area. Oligos provide ~250 bp of coding sequence given a 300-bp oligo with PCR and cloning adaptors. The <t>synthesis</t> cost per gene is shown above the distribution using <t>Twist</t> <t>Bioscience</t> prices for <t>fragment</t> synthesis. Structures are from RCSB Molecule of the Month articles and use the following structure codes from left to right: 1GFL, 4EYL, 5XH3, 1NW9, 1W0E, 2D1S, 1ASZ, and 4OO8. USD, US dollar. ( B ) OMEGA design and assembly workflow. Users provide a list of codon-optimized sequences that OMEGA randomly sorts into N subassembly pools. For each subpool, OMEGA designs fragments that use high-fidelity GG sites and ensures that all pieces are within oligo size constraints. Subassembly pools are separately amplified and assembled into full-length gene products in parallel, after which assembly products are combined to recover the designed library. ( C ) Effect of sequence identity on assembly fidelity. Average predicted assembly fidelity for subpools containing sequences with 50, ~88, or 100% pairwise identity. Each condition was tested using assemblies of 4 or 12 fragments and subpools containing 50 or 70 GG sites. ( D ) Effect of GC content on assembly fidelity. Average predicted assembly fidelity for synthetic sequences with GC content ranging from 20 to 80%. Assemblies were tested for both 4- and 12-fragment constructs and across varied sequence identities. ( E ) Assembly fidelity of repetitive sequences. Average predicted assembly fidelity for DARPin sequences composed of five ankyrin repeats, using 70 GG sites per subpool and varying sequence identities. Mean fidelities across all subpools differed by less than 1%.
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( A ) Distribution of protein lengths for all entries in Swiss-Prot. Most proteins are too large to encode on single oligos, represented as the shaded gray area. Oligos provide ~250 bp of coding sequence given a 300-bp oligo with PCR and cloning adaptors. The <t>synthesis</t> cost per gene is shown above the distribution using <t>Twist</t> <t>Bioscience</t> prices for <t>fragment</t> synthesis. Structures are from RCSB Molecule of the Month articles and use the following structure codes from left to right: 1GFL, 4EYL, 5XH3, 1NW9, 1W0E, 2D1S, 1ASZ, and 4OO8. USD, US dollar. ( B ) OMEGA design and assembly workflow. Users provide a list of codon-optimized sequences that OMEGA randomly sorts into N subassembly pools. For each subpool, OMEGA designs fragments that use high-fidelity GG sites and ensures that all pieces are within oligo size constraints. Subassembly pools are separately amplified and assembled into full-length gene products in parallel, after which assembly products are combined to recover the designed library. ( C ) Effect of sequence identity on assembly fidelity. Average predicted assembly fidelity for subpools containing sequences with 50, ~88, or 100% pairwise identity. Each condition was tested using assemblies of 4 or 12 fragments and subpools containing 50 or 70 GG sites. ( D ) Effect of GC content on assembly fidelity. Average predicted assembly fidelity for synthetic sequences with GC content ranging from 20 to 80%. Assemblies were tested for both 4- and 12-fragment constructs and across varied sequence identities. ( E ) Assembly fidelity of repetitive sequences. Average predicted assembly fidelity for DARPin sequences composed of five ankyrin repeats, using 70 GG sites per subpool and varying sequence identities. Mean fidelities across all subpools differed by less than 1%.
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( A ) Distribution of protein lengths for all entries in Swiss-Prot. Most proteins are too large to encode on single oligos, represented as the shaded gray area. Oligos provide ~250 bp of coding sequence given a 300-bp oligo with PCR and cloning adaptors. The <t>synthesis</t> cost per gene is shown above the distribution using <t>Twist</t> <t>Bioscience</t> prices for <t>fragment</t> synthesis. Structures are from RCSB Molecule of the Month articles and use the following structure codes from left to right: 1GFL, 4EYL, 5XH3, 1NW9, 1W0E, 2D1S, 1ASZ, and 4OO8. USD, US dollar. ( B ) OMEGA design and assembly workflow. Users provide a list of codon-optimized sequences that OMEGA randomly sorts into N subassembly pools. For each subpool, OMEGA designs fragments that use high-fidelity GG sites and ensures that all pieces are within oligo size constraints. Subassembly pools are separately amplified and assembled into full-length gene products in parallel, after which assembly products are combined to recover the designed library. ( C ) Effect of sequence identity on assembly fidelity. Average predicted assembly fidelity for subpools containing sequences with 50, ~88, or 100% pairwise identity. Each condition was tested using assemblies of 4 or 12 fragments and subpools containing 50 or 70 GG sites. ( D ) Effect of GC content on assembly fidelity. Average predicted assembly fidelity for synthetic sequences with GC content ranging from 20 to 80%. Assemblies were tested for both 4- and 12-fragment constructs and across varied sequence identities. ( E ) Assembly fidelity of repetitive sequences. Average predicted assembly fidelity for DARPin sequences composed of five ankyrin repeats, using 70 GG sites per subpool and varying sequence identities. Mean fidelities across all subpools differed by less than 1%.
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( A ) Distribution of protein lengths for all entries in Swiss-Prot. Most proteins are too large to encode on single oligos, represented as the shaded gray area. Oligos provide ~250 bp of coding sequence given a 300-bp oligo with PCR and cloning adaptors. The <t>synthesis</t> cost per gene is shown above the distribution using <t>Twist</t> <t>Bioscience</t> prices for <t>fragment</t> synthesis. Structures are from RCSB Molecule of the Month articles and use the following structure codes from left to right: 1GFL, 4EYL, 5XH3, 1NW9, 1W0E, 2D1S, 1ASZ, and 4OO8. USD, US dollar. ( B ) OMEGA design and assembly workflow. Users provide a list of codon-optimized sequences that OMEGA randomly sorts into N subassembly pools. For each subpool, OMEGA designs fragments that use high-fidelity GG sites and ensures that all pieces are within oligo size constraints. Subassembly pools are separately amplified and assembled into full-length gene products in parallel, after which assembly products are combined to recover the designed library. ( C ) Effect of sequence identity on assembly fidelity. Average predicted assembly fidelity for subpools containing sequences with 50, ~88, or 100% pairwise identity. Each condition was tested using assemblies of 4 or 12 fragments and subpools containing 50 or 70 GG sites. ( D ) Effect of GC content on assembly fidelity. Average predicted assembly fidelity for synthetic sequences with GC content ranging from 20 to 80%. Assemblies were tested for both 4- and 12-fragment constructs and across varied sequence identities. ( E ) Assembly fidelity of repetitive sequences. Average predicted assembly fidelity for DARPin sequences composed of five ankyrin repeats, using 70 GG sites per subpool and varying sequence identities. Mean fidelities across all subpools differed by less than 1%.
Resource Source Identifier Oligonucleotides Custom Oligonucleotide Pool Twist Bioscience N A Readout Probes Idt Dna N A Critical, supplied by Twist Bioscience, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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( A ) Distribution of protein lengths for all entries in Swiss-Prot. Most proteins are too large to encode on single oligos, represented as the shaded gray area. Oligos provide ~250 bp of coding sequence given a 300-bp oligo with PCR and cloning adaptors. The synthesis cost per gene is shown above the distribution using Twist Bioscience prices for fragment synthesis. Structures are from RCSB Molecule of the Month articles and use the following structure codes from left to right: 1GFL, 4EYL, 5XH3, 1NW9, 1W0E, 2D1S, 1ASZ, and 4OO8. USD, US dollar. ( B ) OMEGA design and assembly workflow. Users provide a list of codon-optimized sequences that OMEGA randomly sorts into N subassembly pools. For each subpool, OMEGA designs fragments that use high-fidelity GG sites and ensures that all pieces are within oligo size constraints. Subassembly pools are separately amplified and assembled into full-length gene products in parallel, after which assembly products are combined to recover the designed library. ( C ) Effect of sequence identity on assembly fidelity. Average predicted assembly fidelity for subpools containing sequences with 50, ~88, or 100% pairwise identity. Each condition was tested using assemblies of 4 or 12 fragments and subpools containing 50 or 70 GG sites. ( D ) Effect of GC content on assembly fidelity. Average predicted assembly fidelity for synthetic sequences with GC content ranging from 20 to 80%. Assemblies were tested for both 4- and 12-fragment constructs and across varied sequence identities. ( E ) Assembly fidelity of repetitive sequences. Average predicted assembly fidelity for DARPin sequences composed of five ankyrin repeats, using 70 GG sites per subpool and varying sequence identities. Mean fidelities across all subpools differed by less than 1%.

Journal: Science Advances

Article Title: Scalable and cost-efficient custom gene library assembly from oligopools

doi: 10.1126/sciadv.ady2279

Figure Lengend Snippet: ( A ) Distribution of protein lengths for all entries in Swiss-Prot. Most proteins are too large to encode on single oligos, represented as the shaded gray area. Oligos provide ~250 bp of coding sequence given a 300-bp oligo with PCR and cloning adaptors. The synthesis cost per gene is shown above the distribution using Twist Bioscience prices for fragment synthesis. Structures are from RCSB Molecule of the Month articles and use the following structure codes from left to right: 1GFL, 4EYL, 5XH3, 1NW9, 1W0E, 2D1S, 1ASZ, and 4OO8. USD, US dollar. ( B ) OMEGA design and assembly workflow. Users provide a list of codon-optimized sequences that OMEGA randomly sorts into N subassembly pools. For each subpool, OMEGA designs fragments that use high-fidelity GG sites and ensures that all pieces are within oligo size constraints. Subassembly pools are separately amplified and assembled into full-length gene products in parallel, after which assembly products are combined to recover the designed library. ( C ) Effect of sequence identity on assembly fidelity. Average predicted assembly fidelity for subpools containing sequences with 50, ~88, or 100% pairwise identity. Each condition was tested using assemblies of 4 or 12 fragments and subpools containing 50 or 70 GG sites. ( D ) Effect of GC content on assembly fidelity. Average predicted assembly fidelity for synthetic sequences with GC content ranging from 20 to 80%. Assemblies were tested for both 4- and 12-fragment constructs and across varied sequence identities. ( E ) Assembly fidelity of repetitive sequences. Average predicted assembly fidelity for DARPin sequences composed of five ankyrin repeats, using 70 GG sites per subpool and varying sequence identities. Mean fidelities across all subpools differed by less than 1%.

Article Snippet: To compare costs, we modeled assemblies using 20 to 70 GG sites across gene lengths from 0.3 to 2.5 kb and compared them to Twist Bioscience fragment synthesis, which scales nearly linearly at 7 to 9 cents per base up to 5 kb.

Techniques: Sequencing, Cloning, Amplification, Construct