title: From deep sequencing to actual clones.
abstract: The application of deep sequencing to in vitro display technologies has been invaluable for the straightforward analysis of enriched clones. After sequencing in vitro selected populations, clones are binned into identical or similar groups and ordered by abundance, allowing identification of those that are most enriched. However, the greatest strength of deep sequencing is also its greatest weakness: clones are easily identified by their DNA sequences, but are not physically available for testing without a laborious multistep process involving several rounds of polymerization chain reaction (PCR), assembly and cloning. Here, using the isolation of antibody genes from a phage and yeast display selection as an example, we show the power of a rapid and simple inverse PCR-based method to easily isolate clones identified by deep sequencing. Once primers have been received, clone isolation can be carried out in a single day, rather than two days. Furthermore the reduced number of PCRs required will reduce PCR mutations correspondingly. We have observed a 100% success rate in amplifying clones with an abundance as low as 0.5% in a polyclonal population. This approach allows us to obtain full-length clones even when an incomplete sequence is available, and greatly simplifies the subcloning process. Moreover, rarer, but functional clones missed by traditional screening can be easily isolated using this method, and the approach can be extended to any selected library (scFv, cDNA, libraries based on scaffold proteins) where a unique sequence signature for the desired clones of interest is available.
authors.affiliation: New Mexico Consortium, Los Alamos, NM, USA amb@lanl.gov sdangelo@lanl.gov.
authors.forename: Sara
authors.initials: S
authors.lastname: D'Angelo
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USA Present address: Compugen USA, Inc., San Francisco, CA 94080, USA.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USA.
authors.forename: Leslie
authors.initials: L
authors.lastname: Naranjo
authors.affiliation: New Mexico Consortium, Los Alamos, NM, USA.
authors.forename: Fortunato
authors.initials: F
authors.lastname: Ferrara
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USA.
authors.forename: Csaba
authors.initials: C
authors.lastname: Kiss
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USA amb@lanl.gov sdangelo@lanl.gov.
authors.forename: Andrew R M
authors.initials: AR
authors.lastname: Bradbury
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D061505:Cell Surface Display Techniques / Q000379:methods*; D003001:Cloning, Molecular; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D007779:Lactobacillus acidophilus / Q000235:genetics; D008969:Molecular Sequence Data; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies; D015003:Yeasts / Q000235:genetics
chemical_list: D019151:Peptide Library; D057127:Single-Chain Antibodies
keywords: antibody; deep sequencing; inverse PCR; phage display; yeast display