scholarly journals A comparison of unamplified and massively multiplexed PCR amplification for murine antibody repertoire sequencing

2018 ◽  
Vol 1 (1) ◽  
pp. 6-17 ◽  
Author(s):  
Trisha A. Rettig ◽  
Michael J. Pecaut ◽  
Stephen K. Chapes
Keyword(s):  
Pcr Amplification ◽  
Antibody Repertoire ◽  
Multiplexed Pcr ◽  
Repertoire Sequencing ◽  
Murine Antibody

scholarly journals Dual UMIs and Dual Barcodes With Minimal PCR Amplification Removes Artifacts and Acquires Accurate Antibody Repertoire

2021 ◽  
Vol 12 ◽  
Author(s):  
Qilong Wang ◽  
Huikun Zeng ◽  
Yan Zhu ◽  
Minhui Wang ◽  
Yanfang Zhang ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Antibody Repertoire ◽  
Consensus Building ◽  
Chain Reaction ◽  
Single Nucleotide ◽  
False Discovery ◽  
Novel Approach ◽  
Repertoire Sequencing ◽  
Polymerase Chain ◽  
Antibody Repertoires

Antibody repertoire sequencing (Rep-seq) has been widely used to reveal repertoire dynamics and to interrogate antibodies of interest at single nucleotide-level resolution. However, polymerase chain reaction (PCR) amplification introduces extensive artifacts including chimeras and nucleotide errors, leading to false discovery of antibodies and incorrect assessment of somatic hypermutations (SHMs) which subsequently mislead downstream investigations. Here, a novel approach named DUMPArts, which improves the accuracy of antibody repertoires by labeling each sample with dual barcodes and each molecule with dual unique molecular identifiers (UMIs) via minimal PCR amplification to remove artifacts, is developed. Tested by ultra-deep Rep-seq data, DUMPArts removed inter-sample chimeras, which cause artifactual shared clones and constitute approximately 15% of reads in the library, as well as intra-sample chimeras with erroneous SHMs and constituting approximately 20% of the reads, and corrected base errors and amplification biases by consensus building. The removal of these artifacts will provide an accurate assessment of antibody repertoires and benefit related studies, especially mAb discovery and antibody-guided vaccine design.

scholarly journals Accurate and predictive antibody repertoire profiling by molecular amplification fingerprinting

2016 ◽  
Vol 2 (3) ◽  
pp. e1501371 ◽  
Author(s):  
Tarik A. Khan ◽  
Simon Friedensohn ◽  
Arthur R. Gorter de Vries ◽  
Jakub Straszewski ◽  
Hans-Joachim Ruscheweyh ◽  
...  
Keyword(s):  
Somatic Hypermutation ◽  
Diversity Index ◽  
Pcr Amplification ◽  
Antibody Repertoire ◽  
Library Preparation ◽  
Antibody Diversity ◽  
Synthetic Antibody ◽  
Sequencing Errors ◽  
Repertoire Sequencing ◽  
Improved Accuracy

High-throughput antibody repertoire sequencing (Ig-seq) provides quantitative molecular information on humoral immunity. However, Ig-seq is compromised by biases and errors introduced during library preparation and sequencing. By using synthetic antibody spike-in genes, we determined that primer bias from multiplex polymerase chain reaction (PCR) library preparation resulted in antibody frequencies with only 42 to 62% accuracy. Additionally, Ig-seq errors resulted in antibody diversity measurements being overestimated by up to 5000-fold. To rectify this, we developed molecular amplification fingerprinting (MAF), which uses unique molecular identifier (UID) tagging before and during multiplex PCR amplification, which enabled tagging of transcripts while accounting for PCR efficiency. Combined with a bioinformatic pipeline, MAF bias correction led to measurements of antibody frequencies with up to 99% accuracy. We also used MAF to correct PCR and sequencing errors, resulting in enhanced accuracy of full-length antibody diversity measurements, achieving 98 to 100% error correction. Using murine MAF-corrected data, we established a quantitative metric of recent clonal expansion—the intraclonal diversity index—which measures the number of unique transcripts associated with an antibody clone. We used this intraclonal diversity index along with antibody frequencies and somatic hypermutation to build a logistic regression model for prediction of the immunological status of clones. The model was able to predict clonal status with high confidence but only when using MAF error and bias corrected Ig-seq data. Improved accuracy by MAF provides the potential to greatly advance Ig-seq and its utility in immunology and biotechnology.

scholarly journals Accurate Immune Repertoire Sequencing Reveals Malaria Infection Driven Antibody Lineage Diversification in Young Children

2017 ◽  
Author(s):  
Ben S. Wendel ◽  
Chenfeng He ◽  
Mingjuan Qu ◽  
Di Wu ◽  
Stefany M. Hernandez ◽  
...  
Keyword(s):  
B Cells ◽  
Somatic Hypermutation ◽  
Antibody Repertoire ◽  
Infants And Toddlers ◽  
High Coverage ◽  
Sequence Composition ◽  
Age Related ◽  
Sequencing Method ◽  
Repertoire Sequencing ◽  
Lineage Diversification

ABSTRACTAccurately measuring antibody repertoire sequence composition in a small amount of blood is challenging yet important to the understanding of the repertoire response to infections and vaccinations. Here, we describe an accurate and high-coverage repertoire sequencing method, MIDCIRS, which uses as few as 1,000 naïve B cells. Using it, we studied age-related antibody repertoire development and diversification before and during acute malaria in infants (< 12 months old) and toddlers (12 – 47 months old) with 4-8 ml of blood draws. Unexpectedly, we discovered high levels of somatic hypermutation (SHM) in infants as young as three months old. Antibody clonal lineage analysis revealed that both infants and toddlers increase SHM levels upon infection and memory B cells isolated from pre-malaria samples in malaria-experienced individuals continue to induce SHMs upon malaria rechallenge. These results highlight the vast potential of antibody repertoire diversification in infants and toddlers that has not been realized previously.

scholarly journals Knowledge-based antibody repertoire simulation, a novel allele detection tool evaluation and application

2021 ◽  
Author(s):  
Xiujia Yang ◽  
Yan Zhu ◽  
Huikun Zeng ◽  
Sen Chen ◽  
Junjie Guan ◽  
...  
Keyword(s):  
Humoral Immunity ◽  
Antibody Repertoire ◽  
Detailed Knowledge ◽  
Allele Detection ◽  
Full Spectrum ◽  
Knowledge Based ◽  
Gene Usage ◽  
Repertoire Sequencing ◽  
Novel Alleles ◽  
Novel Allele

Detailed knowledge of the diverse immunoglobulin germline genes is critical for the study of humoral immunity. Hundreds of alleles have been discovered by analyzing antibody repertoire sequencing (Rep-seq or Ig-seq) data via multiple novel allele detection tools (NADTs). However, the performance of these NADTs through antibody sequences with intrinsic somatic hypermutations (SHMs) is unclear. Here, we developed a tool to simulate repertoires by integrating the full spectrum features of an antibody repertoire such as germline gene usage, junctional modification, position-specific SHM and clonal expansion based on 2152 high-quality datasets. We then systematically evaluated these NADTs using both simulated and genuine Ig-seq datasets. Finally, we applied these NADTs to 687 Ig-seq datasets and identified 43 novel alleles using defined criteria. Twenty-five alleles were validated through findings of other sources. In addition to the novel alleles detected, our simulation tool, the results of our comparison, and the streamline of this process may benefit further humoral immunity studies via Ig-seq.

scholarly journals Multi-Donor Longitudinal Antibody Repertoire Sequencing Reveals the Existence of Public Antibody Clonotypes in HIV-1 Infection

2018 ◽  
Vol 23 (6) ◽  
pp. 845-854.e6 ◽  
Author(s):  
Ian Setliff ◽  
Wyatt J. McDonnell ◽  
Nagarajan Raju ◽  
Robin G. Bombardi ◽  
Amyn A. Murji ◽  
...  
Keyword(s):  
Antibody Repertoire ◽  
Repertoire Sequencing ◽  
Hiv 1

scholarly journals Simple paired heavy- and light-chain antibody repertoire sequencing using endoplasmic reticulum microsomes

2018 ◽  
Vol 10 (1) ◽  
Author(s):  
Praneeth Reddy Devulapally ◽  
Jörg Bürger ◽  
Thorsten Mielke ◽  
Zoltán Konthur ◽  
Hans Lehrach ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Light Chain ◽  
Antibody Repertoire ◽  
Repertoire Sequencing ◽  
Light Chain Antibody

Population genetics of 10 Y-chromosomal STR loci in Japanese using multiplexed PCR amplification kits, Y-PLEX™6 and Y-PLEX™5

2004 ◽  
Vol 1261 ◽  
pp. 307-309 ◽  
Author(s):  
A. Kido ◽  
M. Hara ◽  
H. Kameyama ◽  
Y. Yamamoto ◽  
R. Susukida ◽  
...  
Keyword(s):  
Population Genetics ◽  
Pcr Amplification ◽  
Str Loci ◽  
Multiplexed Pcr

scholarly journals Massively scalable genetic analysis of antibody repertoires

2018 ◽  
Author(s):  
Bryan Briney ◽  
Dennis R. Burton
Keyword(s):  
Genetic Analysis ◽  
Sequence Data ◽  
Read Length ◽  
Antibody Repertoire ◽  
Link Type ◽  
Repertoire Sequencing ◽  
Antibody Sequence ◽  
Downstream Analysis ◽  
Sequencing Platforms ◽  
Computational Resources

AbstractWith technical breakthroughs in the throughput and read-length of next-generation sequencing platforms, antibody repertoire sequencing is becoming an increasingly important tool for detailed characterization of the immune response. There is a need for open, scalable software for the genetic analysis of repertoire-scale antibody sequence data. To address this gap, we have developed the ab[x] package of software tools. There are three core components of the ab[x] toolkit, all of which are freely available: abcloud (github.com/briney/abcloud) for deployment and management of computational resources on Amazon’s Elastic Compute Cloud; abstar (github.com/briney/abstar) for pre-processing, germline gene assignment and primary annotation of antibody sequence data; and abutils (github.com/briney/abutils), which provides utilities for interactive downstream analysis of antibody repertoire data.

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