title: High-quality full-length immunoglobulin profiling with unique molecular barcoding.
abstract: High-throughput sequencing analysis of hypermutating immunoglobulin (IG) repertoires remains a challenging task. Here we present a robust protocol for the full-length profiling of human and mouse IG repertoires. This protocol uses unique molecular identifiers (UMIs) introduced in the course of cDNA synthesis to control bottlenecks and to eliminate PCR and sequencing errors. Using asymmetric 400+100-nt paired-end Illumina sequencing and UMI-based assembly with the new version of the MIGEC software, the protocol allows up to 750-nt lengths to be sequenced in an almost error-free manner. This sequencing approach should also be applicable to various tasks beyond immune repertoire studies. In IG profiling, the achieved length of high-quality sequence covers the variable region of even the longest chains, along with the fragment of a constant region carrying information on the antibody isotype. The whole protocol, including preparation of cells and libraries, sequencing and data analysis, takes 5 to 6 d.
authors.affiliation: Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Moscow, Russia.|Center of Molecular Medicine, CEITEC, Masaryk University, Brno, Czech Republic.|Pirogov Russian National Research Medical University, Moscow, Russia.
authors.forename: M A
authors.initials: MA
authors.lastname: Turchaninova
authors.affiliation: Center of Molecular Medicine, CEITEC, Masaryk University, Brno, Czech Republic.
authors.forename: A
authors.initials: A
authors.lastname: Davydov
authors.affiliation: Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Moscow, Russia.|Pirogov Russian National Research Medical University, Moscow, Russia.
authors.forename: O V
authors.initials: OV
authors.lastname: Britanova
authors.affiliation: Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Moscow, Russia.|Pirogov Russian National Research Medical University, Moscow, Russia.
authors.forename: M
authors.identifier: 0000-0001-7826-7942
authors.initials: M
authors.lastname: Shugay
authors.affiliation: Center of Molecular Medicine, CEITEC, Masaryk University, Brno, Czech Republic.
authors.forename: V
authors.initials: V
authors.lastname: Bikos
authors.affiliation: Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Moscow, Russia.|Center of Molecular Medicine, CEITEC, Masaryk University, Brno, Czech Republic.|Pirogov Russian National Research Medical University, Moscow, Russia.
authors.forename: E S
authors.initials: ES
authors.lastname: Egorov
authors.affiliation: Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Moscow, Russia.
authors.forename: V I
authors.initials: VI
authors.lastname: Kirgizova
authors.affiliation: Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Moscow, Russia.
authors.forename: E M
authors.initials: EM
authors.lastname: Merzlyak
authors.affiliation: Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Moscow, Russia.|Pirogov Russian National Research Medical University, Moscow, Russia.
authors.forename: D B
authors.initials: DB
authors.lastname: Staroverov
authors.affiliation: Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Moscow, Russia.|Pirogov Russian National Research Medical University, Moscow, Russia.
authors.forename: D A
authors.identifier: 0000-0002-8484-6067
authors.initials: DA
authors.lastname: Bolotin
authors.affiliation: Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Moscow, Russia.|Center of Molecular Medicine, CEITEC, Masaryk University, Brno, Czech Republic.
authors.forename: I Z
authors.initials: IZ
authors.lastname: Mamedov
authors.affiliation: Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Moscow, Russia.|Center of Molecular Medicine, CEITEC, Masaryk University, Brno, Czech Republic.|Pirogov Russian National Research Medical University, Moscow, Russia.
authors.forename: M
authors.initials: M
authors.lastname: Izraelson
authors.affiliation: Pirogov Russian National Research Medical University, Moscow, Russia.
authors.forename: M D
authors.initials: MD
authors.lastname: Logacheva
authors.affiliation: Pirogov Russian National Research Medical University, Moscow, Russia.
authors.forename: O
authors.initials: O
authors.lastname: Kladova
authors.affiliation: Center of Molecular Medicine, CEITEC, Masaryk University, Brno, Czech Republic.
authors.forename: K
authors.initials: K
authors.lastname: Plevova
authors.affiliation: Center of Molecular Medicine, CEITEC, Masaryk University, Brno, Czech Republic.
authors.forename: S
authors.initials: S
authors.lastname: Pospisilova
authors.affiliation: Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Moscow, Russia.|Center of Molecular Medicine, CEITEC, Masaryk University, Brno, Czech Republic.|Pirogov Russian National Research Medical University, Moscow, Russia.
authors.forename: D M
authors.identifier: 0000-0003-0430-790X
authors.initials: DM
authors.lastname: Chudakov
mesh_terms: D000818:Animals; D001483:Base Sequence; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007147:Immunoglobulin Light Chains / Q000235:genetics*; D051379:Mice; D009154:Mutation; D011786:Quality Control; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains