Assessment of antibody library diversity through next generation sequencing and technical error compensation

Antibody libraries are important resources to derive antibodies to be used for a wide range of applications, from structural and functional studies to intracellular protein interference studies to developing new diagnostics and therapeutics. Whatever the goal, the key parameter for an antibody library is its complexity (also known as diversity), i.e. the number of distinct elements in the collection, which directly reflects the probability of finding in the library an antibody against a given antigen, of sufficiently high affinity. Quantitative evaluation of antibody library complexity and quality has been for a long time inadequately addressed, due to the high similarity and length of the sequences of the library. Complexity was usually inferred by the transformation efficiency and tested either by fingerprinting and/or sequencing of a few hundred random library elements. Inferring complexity from such a small sampling is, however, very rudimental and gives limited information about the real diversity, because complexity does not scale linearly with sample size. Next-generation sequencing (NGS) has opened new ways to tackle the antibody library complexity quality assessment. However, much remains to be done to fully exploit the potential of NGS for the quantitative analysis of antibody repertoires and to overcome current limitations. To obtain a more reliable antibody library complexity estimate here we show a new, PCR-free, NGS approach to sequence antibody libraries on Illumina platform, coupled to a new bioinformatic analysis and software (Diversity Estimator of Antibody Library, DEAL) that allows to reliably estimate the complexity, taking in consideration the sequencing error.

[1]  Victor Greiff,et al.  Quantitative assessment of the robustness of next-generation sequencing of antibody variable gene repertoires from immunized mice , 2014, BMC Immunology.

[2]  T. Rabbitts,et al.  Single domain intracellular antibodies: a minimal fragment for direct in vivo selection of antigen-specific intrabodies. , 2003, Journal of molecular biology.

[3]  Eric T. Boder,et al.  Yeast surface display for screening combinatorial polypeptide libraries , 1997, Nature Biotechnology.

[4]  Stephen R. Quake,et al.  Genetic measurement of memory B-cell recall using antibody repertoire sequencing , 2013, Proceedings of the National Academy of Sciences.

[5]  Björn Usadel,et al.  Trimmomatic: a flexible trimmer for Illumina sequence data , 2014, Bioinform..

[6]  T. Sirinarumitr,et al.  Efficient amplification of light and heavy chain variable regions and construction of a non-immune phage scFv library , 2010, Molecular Biology Reports.

[7]  Jan Terje Kvaløy,et al.  Error propagation in relative real-time reverse transcription polymerase chain reaction quantification models: the balance between accuracy and precision. , 2006, Analytical biochemistry.

[8]  T. Rabbitts,et al.  De novo production of diverse intracellular antibody libraries. , 2003, Nucleic acids research.

[9]  A. Plückthun,et al.  In vitro selection and evolution of functional proteins by using ribosome display. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[10]  N. Fischer,et al.  Sequencing antibody repertoires: the next generation. , 2011, mAbs.

[11]  N. Kyrpides,et al.  Direct Comparisons of Illumina vs. Roche 454 Sequencing Technologies on the Same Microbial Community DNA Sample , 2012, PloS one.

[12]  A. Garen,et al.  A melanoma-specific VH antibody cloned from a fusion phage library of a vaccinated melanoma patient. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[13]  G. Oster,et al.  Theoretical studies of clonal selection: minimal antibody repertoire size and reliability of self-non-self discrimination. , 1979, Journal of theoretical biology.

[14]  Jan Berka,et al.  Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire , 2009, Proceedings of the National Academy of Sciences.

[15]  Kin-Fan Au,et al.  PacBio Sequencing and Its Applications , 2015, Genom. Proteom. Bioinform..

[16]  H R Hoogenboom,et al.  By-passing immunization. Human antibodies from V-gene libraries displayed on phage. , 1991, Journal of molecular biology.

[17]  William Hyde Wollaston,et al.  I. The Croonian Lecture , 1810, Philosophical Transactions of the Royal Society of London.

[18]  L. Wyns,et al.  Selection and identification of single domain antibody fragments from camel heavy‐chain antibodies , 1997, FEBS letters.

[19]  S. Quake,et al.  The promise and challenge of high-throughput sequencing of the antibody repertoire , 2014, Nature Biotechnology.

[20]  T. Rabbitts,et al.  Protocol for the selection of single-domain antibody fragments by third generation intracellular antibody capture , 2010, Nature Protocols.

[21]  M. Taussig,et al.  Antibody-ribosome-mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites. , 1997, Nucleic acids research.

[22]  J. Marks,et al.  PCR cloning of human immunoglobulin genes. , 2004, Methods in molecular biology.

[23]  T. Rabbitts,et al.  Selection of antibodies for intracellular function using a two-hybrid in vivo system. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[24]  M. Quondam,et al.  The intracellular antibody capture technology: towards the high-throughput selection of functional intracellular antibodies for target validation. , 2004, Methods.

[25]  I. Mårtensson,et al.  Transcription of productive and nonproductive VDJ‐recombined alleles after IgH allelic exclusion , 2007, The EMBO journal.

[26]  César Milstein,et al.  Man-made antibodies , 1991, Nature.

[27]  A. Chao,et al.  Estimating the Number of Classes via Sample Coverage , 1992 .

[28]  Mikhail Shugay,et al.  Towards error-free profiling of immune repertoires , 2014, Nature Methods.

[29]  R. White,et al.  High-Throughput Sequencing of the Zebrafish Antibody Repertoire , 2009, Science.

[30]  A. Cattaneo,et al.  Intracellular antibodies for proteomics☆ , 2004, Journal of Immunological Methods.

[31]  R. Fuller,et al.  Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences. , 2011, Cold Spring Harbor protocols.

[32]  Jiajie Zhang,et al.  PEAR: a fast and accurate Illumina Paired-End reAd mergeR , 2013, Bioinform..

[33]  J. Young,et al.  Selection of specific phage-display antibodies using libraries derived from chicken immunoglobulin genes. , 1995, Journal of immunological methods.

[34]  James L. Zehnder,et al.  High-throughput VDJ sequencing for quantification of minimal residual disease in chronic lymphocytic leukemia and immune reconstitution assessment , 2011, Proceedings of the National Academy of Sciences.

[35]  C. Barbas,et al.  Recombinant rabbit Fab with binding activity to type-1 plasminogen activator inhibitor derived from a phage-display library against human alpha-granules. , 1996, Gene.

[36]  I. Arisi,et al.  Post-translational selective intracellular silencing of acetylated proteins with de novo selected intrabodies , 2017, Nature Methods.

[37]  C. Milstein.  The Croonian Lecture, 1989 Antibodies: a paradigm for the biology of molecular recognition , 1990, Proceedings of the Royal Society of London. B. Biological Sciences.

[38]  M. Schlissel,et al.  Allelic exclusion of immunoglobulin genes: models and mechanisms , 2010, Immunological reviews.

[39]  H R Hoogenboom,et al.  Antibody phage display technology and its applications. , 1998, Immunotechnology : an international journal of immunological engineering.

[40]  A. Cattaneo,et al.  Direct in vivo intracellular selection of conformation-sensitive antibody domains targeting Alzheimer's amyloid-beta oligomers. , 2009, Journal of molecular biology.

[41]  Emese Meglécz,et al.  Accuracy and quality assessment of 454 GS-FLX Titanium pyrosequencing , 2011, BMC Genomics.

[42]  George Georgiou,et al.  In-depth determination and analysis of the human paired heavy- and light-chain antibody repertoire , 2014, Nature Medicine.

[43]  George Georgiou,et al.  High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire , 2013, Nature Biotechnology.

[44]  K. Ansell,et al.  Isolation of tumor cell‐specific single‐chain Fv from immunized mice using phage‐antibody libraries and the re‐construction of whole antibodies from these antibody fragments , 1994, European journal of immunology.

[45]  S. Reddy,et al.  Deep sequencing in library selection projects: what insight does it bring? , 2015, Current opinion in structural biology.