www.nature.com/articles/s41...
Excited to have this collaboration from #QVEU and @rongellerlab.bsky.social lab out in the world! After publishing our own #DeepMutationalScanning studies on two related viruses, we asked what we learn from comparisons of such datasets. The answer is a lot!
Strategy to generate a DMS plasmid library for Your Favorite Gene (YFG) using short, degenerate libraries. 1. Segmentation of YFG into sub-fragments, each fragment corresponding to a DNA region to be synthesized. The same approach can be applied to promoter and terminator regions, if desired. 2. Example of a pool of degenerate oligonucleotides (oPool) derived from one YFG fragment associated with DNA barcodes. Each oPool contains: (i) ~40 bp of homology upstream of the YFG fragment of interest, (ii) the YFG fragment sequence with a single NNK codon, (iii) BsaI cloning sites, (iv) a DNA barcode composed of codon-position specific regions and six degenerate nucleotides (N), and (v) a conserved i7 primer binding site (PBS_i7) present in all oPools and used for rapid and efficient sequencing library preparation. Current oligonucleotide synthesis technologies allow for a total of nine degenerate positions per fragment: three are used for the degenerate codon (NNK), and six for the barcode. A complete list of all oPool sequences and their detailed composition is provided in S1 Table. 3. Protocol for constructing YFG DMS plasmid library from oPools using two cloning steps that maintain the physical barcode-mutation association. The libraries of oPools are cloned into the plasmid template by Gibson cloning. Following this step, for each fragment, a necessary short-read sequencing using PBS_i5 (included in the 5′ sequencing primer) and PBS_i7 is performed to associate each barcode with its corresponding mutation and to assess both barcode diversity per mutation and mutation coverage for the whole fragment. The ultimate step consists in Golden Gate cloning of the missing 3′ gene fragment between the degenerate fragment and the barcode. An additional short-read sequencing step of the barcodes can be performed to make sure that coverage and diversity have been maintained. Figure created in BioRender.
#DeepMutationalScanning (DMS) experiments are limited by gene size due to library complexity & costs. @christianlandry.bsky.social &co develop an efficient & cost-effective barcoded cloning strategy for plasmid-based DMS libraries that enables study of large genes @plosbiology.org 🧪 plos.io/4abhyUf
Strategy to generate a DMS plasmid library for Your Favorite Gene (YFG) using short, degenerate libraries. 1. Segmentation of YFG into sub-fragments, each fragment corresponding to a DNA region to be synthesized. The same approach can be applied to promoter and terminator regions, if desired. 2. Example of a pool of degenerate oligonucleotides (oPool) derived from one YFG fragment associated with DNA barcodes. Each oPool contains: (i) ~40 bp of homology upstream of the YFG fragment of interest, (ii) the YFG fragment sequence with a single NNK codon, (iii) BsaI cloning sites, (iv) a DNA barcode composed of codon-position specific regions and six degenerate nucleotides (N), and (v) a conserved i7 primer binding site (PBS_i7) present in all oPools and used for rapid and efficient sequencing library preparation. Current oligonucleotide synthesis technologies allow for a total of nine degenerate positions per fragment: three are used for the degenerate codon (NNK), and six for the barcode. A complete list of all oPool sequences and their detailed composition is provided in S1 Table. 3. Protocol for constructing YFG DMS plasmid library from oPools using two cloning steps that maintain the physical barcode-mutation association. The libraries of oPools are cloned into the plasmid template by Gibson cloning. Following this step, for each fragment, a necessary short-read sequencing using PBS_i5 (included in the 5′ sequencing primer) and PBS_i7 is performed to associate each barcode with its corresponding mutation and to assess both barcode diversity per mutation and mutation coverage for the whole fragment. The ultimate step consists in Golden Gate cloning of the missing 3′ gene fragment between the degenerate fragment and the barcode. An additional short-read sequencing step of the barcodes can be performed to make sure that coverage and diversity have been maintained. Figure created in BioRender.
#DeepMutationalScanning (DMS) experiments are limited by gene size due to library complexity & costs. @christianlandry.bsky.social &co develop an efficient & cost-effective barcoded cloning strategy for plasmid-based DMS libraries that enables study of large genes @plosbiology.org 🧪 plos.io/4abhyUf
Strategy to generate a DMS plasmid library for Your Favorite Gene (YFG) using short, degenerate libraries. 1. Segmentation of YFG into sub-fragments, each fragment corresponding to a DNA region to be synthesized. The same approach can be applied to promoter and terminator regions, if desired. 2. Example of a pool of degenerate oligonucleotides (oPool) derived from one YFG fragment associated with DNA barcodes. Each oPool contains: (i) ~40 bp of homology upstream of the YFG fragment of interest, (ii) the YFG fragment sequence with a single NNK codon, (iii) BsaI cloning sites, (iv) a DNA barcode composed of codon-position specific regions and six degenerate nucleotides (N), and (v) a conserved i7 primer binding site (PBS_i7) present in all oPools and used for rapid and efficient sequencing library preparation. Current oligonucleotide synthesis technologies allow for a total of nine degenerate positions per fragment: three are used for the degenerate codon (NNK), and six for the barcode. A complete list of all oPool sequences and their detailed composition is provided in S1 Table. 3. Protocol for constructing YFG DMS plasmid library from oPools using two cloning steps that maintain the physical barcode-mutation association. The libraries of oPools are cloned into the plasmid template by Gibson cloning. Following this step, for each fragment, a necessary short-read sequencing using PBS_i5 (included in the 5′ sequencing primer) and PBS_i7 is performed to associate each barcode with its corresponding mutation and to assess both barcode diversity per mutation and mutation coverage for the whole fragment. The ultimate step consists in Golden Gate cloning of the missing 3′ gene fragment between the degenerate fragment and the barcode. An additional short-read sequencing step of the barcodes can be performed to make sure that coverage and diversity have been maintained. Figure created in BioRender.
#DeepMutationalScanning (DMS) experiments are limited by gene size due to library complexity & costs. @christianlandry.bsky.social &co develop an efficient & cost-effective barcoded cloning strategy for plasmid-based DMS libraries that enables study of large genes @plosbiology.org 🧪 plos.io/4abhyUf
Critical Assessment of Genome Interpretation (CAGI) challenges are live! 🧬💻
genomeinterpretation.org/cagi7-challe...
A total of 12 challenges have launched Including #DeepMutationalScanning Challenges
⏰ Deadlines approaching! #CAGI #GenomeInterpretation #VariantEffect
Groundbreaking method dmSLAY improves antimicrobial peptide effectiveness and safety 🔬🧬✨ www.news-medical.net/news/2024080... #Antimicrobial #BioEngineering #MachineLearning #DeepMutationalScanning #SyntheticBiology #Peptides #Microbiology #BioTech @natureportfolio.bsky.social
