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Quantitative assessment of the robustness of next-generation sequencing of antibody variable gene repertoires from immunized mice

Overview of attention for article published in BMC Immunology, October 2014
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  • High Attention Score compared to outputs of the same age and source (87th percentile)

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1 tweeter
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1 patent

Citations

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Title
Quantitative assessment of the robustness of next-generation sequencing of antibody variable gene repertoires from immunized mice
Published in
BMC Immunology, October 2014
DOI 10.1186/s12865-014-0040-5
Pubmed ID
Authors

Victor Greiff, Ulrike Menzel, Ulrike Haessler, Skylar C Cook, Simon Friedensohn, Tarik A Khan, Mark Pogson, Ina Hellmann, Sai T Reddy

Abstract

BackgroundNext-generation sequencing (NGS) of antibody variable regions has emerged as a powerful tool in systems immunology by providing quantitative molecular information on polyclonal humoral immune responses. Reproducible and robust information on antibody repertoires is valuable for basic and applied immunology studies: thus, it is essential to establish the reliability of antibody NGS data.ResultsWe isolated RNA from antibody-secreting cells (ASCs) from either 1 mouse or a pool of 9 immunized mice in order to simulate both normal and high diversity populations. Next, we prepared three technical replicates of antibody libraries by RT-PCR from each diversity scenario, which were sequenced using the Illumina MiSeq platform resulting in >106 250 bp paired-end reads per replicate. We then assessed the robustness of antibody repertoire data based on clonal identification defined by amino acid sequence of either full-length VDJ region or the complementarity determining region 3 (CDR3). Leveraging modeling approaches adapted from mathematical ecology, we found that in either diversity scenario both CDR3 and VDJ detection nears completeness indicating deep coverage of ASC repertoires. Additionally, we defined reliability thresholds for accurate quantification and ranking of CDR3s and VDJs. Importantly, we show that both factors¿(i) replicate sequencing and (ii) sequencing depth¿are crucial for robust CDR3 and VDJ detection and ranking.ConclusionsIn summary, we established widely applicable experimental and computational guidelines for robust antibody NGS and analysis, which will help advance systems immunology studies related to the quantitative profiling of antibody responses following infection and vaccination.

Twitter Demographics

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Mendeley readers

The data shown below were compiled from readership statistics for 149 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
United States 2 1%
United Kingdom 1 <1%
Russia 1 <1%
Unknown 145 97%

Demographic breakdown

Readers by professional status Count As %
Researcher 51 34%
Student > Ph. D. Student 33 22%
Student > Master 12 8%
Student > Bachelor 12 8%
Other 10 7%
Other 16 11%
Unknown 15 10%
Readers by discipline Count As %
Agricultural and Biological Sciences 53 36%
Biochemistry, Genetics and Molecular Biology 38 26%
Immunology and Microbiology 22 15%
Medicine and Dentistry 5 3%
Pharmacology, Toxicology and Pharmaceutical Science 3 2%
Other 10 7%
Unknown 18 12%

Attention Score in Context

This research output has an Altmetric Attention Score of 4. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 23 January 2020.
All research outputs
#4,966,020
of 16,652,557 outputs
Outputs from BMC Immunology
#86
of 480 outputs
Outputs of similar age
#66,466
of 235,907 outputs
Outputs of similar age from BMC Immunology
#3
of 33 outputs
Altmetric has tracked 16,652,557 research outputs across all sources so far. This one has received more attention than most of these and is in the 69th percentile.
So far Altmetric has tracked 480 research outputs from this source. They receive a mean Attention Score of 3.2. This one has done well, scoring higher than 81% of its peers.
Older research outputs will score higher simply because they've had more time to accumulate mentions. To account for age we can compare this Altmetric Attention Score to the 235,907 tracked outputs that were published within six weeks on either side of this one in any source. This one has gotten more attention than average, scoring higher than 70% of its contemporaries.
We're also able to compare this research output to 33 others from the same source and published within six weeks on either side of this one. This one has done well, scoring higher than 87% of its contemporaries.