BALDR: a computational pipeline for paired heavy and light chain immunoglobulin reconstruction in single-cell RNA-seq data

Upadhyay, Amit A.; Kauffman, Robert C.; Wolabaugh, Amber N.; Cho, Alice; Patel, Nirav; Reiss, Samantha M.; Havenar-Daughton, Colin; Dawoud, Reem A.; Tharp, Gregory K.; Sanz, Iñaki; Pulendran, Bali; Crotty, Shane; Lee, F. Eun-Hyung; Wrammert, Jens; Bosinger, Steven E.

doi:10.1186/s13073-018-0528-3

Cited by 65 publications

(68 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For library preparation, amplified cDNA is fragmented and appended with dual-indexed barcodes using Illumina Nextera XT DNA Library Prep kits. Sequencing was performed using 101-bp single end reads at Yerkes Genomics Core (http://www.yerkes.emory.edu/nhp_genomics_core/) as previously described (75) on an Illumina HiSeq 3000 at a depth of ∼1,000,000 reads per cell. In total, 127 cells were successfully captured and profiled for single-cell transcriptomic analysis: 25 Mock cells, 68 WNV cells, and 34 WNV (+Ab) cells.…”

Section: Methodsmentioning

confidence: 99%

West Nile virus-inclusive single-cell RNA sequencing reveals heterogeneity in the type I interferon response within single cells

O’Neal

Upadhyay

Wolabaugh

et al. 2018

Preprint

Self Cite

View full text Add to dashboard Cite

232 words; Importance: 122 words; and Text: 4,850 words. 22 ABSTRACT 23West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus of global importance. 24Neuroinvasive WNV infection results in encephalitis and can lead to prolonged 25 neurological impairment or death. Type I interferon (IFN-I) is crucial for promoting 26 antiviral defenses through the induction of antiviral effectors, which function to restrict 27 viral replication and spread. However, our understanding of the antiviral response to 28 WNV infection is mostly derived from analysis of bulk cell populations. It is becoming 29 increasingly apparent that substantial heterogeneity in cellular processes exists among 30 individual cells, even within a seemingly homogenous cell population. Here, we present 31 WNV-inclusive single-cell RNA sequencing (scRNA-seq), an approach to examine the 32 transcriptional variation and viral RNA burden across single cells. We observed that 33 only a few cells within the bulk population displayed robust transcription of IFN-β mRNA, 34 and this did not appear to depend on viral RNA abundance within the same cell. 35 Furthermore, we observed considerable transcriptional heterogeneity in the IFN-I 36 response, with genes displaying high unimodal and bimodal expression patterns. 37 Broadly, IFN-stimulated genes negatively correlated with viral RNA abundance, 38 corresponding with a precipitous decline in expression in cells with high viral RNA levels. 39 Altogether, we demonstrated the feasibility and utility of WNV-inclusive scRNA-seq as a 40 high-throughput technique for single-cell transcriptomics and WNV RNA detection. This 41 approach can be implemented in other models to provide insights into the cellular 42 features of protective immunity and identify novel therapeutic targets. 43 44 45 IMPORTANCE 46 West Nile virus (WNV) is a clinically relevant pathogen responsible for recurrent 47 epidemics of neuroinvasive disease. Type I interferon is essential for promoting an 48 antiviral response against WNV infection; however, it is unclear how heterogeneity in 49 the antiviral response at the single-cell level impacts viral control. Specifically, 50 conventional approaches lack the ability to distinguish differences across cells with 51 varying viral abundance. The significance of our research is to demonstrate a new 52 technique for studying WNV infection at the single-cell level. We discovered extensive 53 variation in antiviral gene expression and viral abundance across cells. This protocol 54 can be applied to primary cells or in vivo models to better understand the underlying 55 cellular heterogeneity following WNV infection for the development of targeted 56 therapeutic strategies. 57 58 59 60 61

show abstract

Section: Methodsmentioning

confidence: 99%

West Nile virus-inclusive single-cell RNA sequencing reveals heterogeneity in the type I interferon response within single cells

O’Neal

Upadhyay

Wolabaugh

et al. 2018

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…To obtain paired heavy and light chain sequences from single cell RNA-Seq data, we used the BALDR pipeline, as previously described (Upadhyay et al, 2018), with the Unfiltered method for rhesus macaques.…”

Section: Star Methodsmentioning

confidence: 99%

Slow delivery immunization enhances HIV neutralizing antibody and germinal center responses via modulation of immunodominance

Cirelli

Carnathan

Nogal

et al. 2018

Preprint

Self Cite

View full text Add to dashboard Cite

35The observation that humans can produce broadly neutralizing antibodies (bnAbs) against HIV-1 has 36 generated enthusiasm about the potential for a bnAb vaccine against HIV-1. Conventional immunization 37 strategies will likely be insufficient for the development of a bnAb HIV vaccine and vaccines to other 38 difficult pathogens, due to the significant immunological hurdles posed, including B cell 39 immunodominance and germinal center (GC) quantity and quality. Using longitudinal lymph node fine 40 needle aspirates, we found that two independent methods of slow delivery immunization of rhesus 41 macaques (RM) resulted in larger GCs, more robust and sustained GC Tfh cell responses, and GC B cells 42 with improved Env-binding, which correlated with the development of ~20 to 30-fold higher titers of tier 43 2 HIV-1 nAbs. Using a new RM genomic immunoglobulin loci reference sequence, we identified 44 differential IgV gene usage between slow delivery immunized and conventional bolus immunized 45 animals. The most immunodominant IgV gene used by conventionally immunized animals was 46 associated with many GC B cell lineages. Ab mapping of those GC B cell specificities demonstrated 47 targeting of an immunodominant non-neutralizing trimer base epitope, while that response was muted 48 in slow delivery immunized animals. Thus, alternative immunization strategies appear to enhance nAb 49 development by altering GCs and modulating immunodominance of non-neutralizing epitopes. 50 51

show abstract

“…The development of single-cell RNA-seq (scRNA-seq) protocols provides a unique tool to identify and characterize cell subsets according to their full transcriptional profile. [23][24][25] Additionally, recent computational tools for reconstruction of antigen receptor sequences from scRNA-seq data [26][27][28][29][30][31][32][33] now allow us to link the transcriptional profile of individual PCs with information about their B-cell receptor (BCR) and thus clonal relatedness.…”

Section: Introductionmentioning

confidence: 99%

Longevity, clonal relationship and transcriptional program of celiac disease-specific plasma cells

Lindeman

Zhou

Eggesbø

et al. 2020

Preprint

View full text Add to dashboard Cite

Disease-specific plasma cells (PCs) reactive with transglutaminase 2 (TG2) or deamidated gluten peptides (DGP) are abundant in celiac disease (CeD) gut lesions. Their contribution toward CeD pathogenesis is unclear. We assessed expression of markers associated with PC longevity in 15 untreated and 26 treated CeD patients in addition to 13 non-CeD controls, and performed RNAsequencing with clonal inference and transcriptomic analysis of 3251 single PCs. We observed antigen-dependent V-gene selection and stereotypic antibodies. Generation of recombinant DGPspecific antibodies revealed a key role of a heavy-chain residue that displays polymorphism, suggesting that immunoglobulin gene polymorphisms may influence CeD-specific antibody responses.We identified transcriptional differences between CeD-specific vs non-disease-specific PCs and between short-lived vs long-lived PCs. The short-lived CD19 + CD45 + phenotype dominated in untreated and short-term-treated CeD, in particular among disease-specific PCs but also in the general PC population. Thus, the disease lesion of untreated CeD is characterized by massive accumulation of short-lived PCs that are not only directed against disease-specific antigens.

show abstract

BALDR: a computational pipeline for paired heavy and light chain immunoglobulin reconstruction in single-cell RNA-seq data

Cited by 65 publications

References 47 publications

West Nile virus-inclusive single-cell RNA sequencing reveals heterogeneity in the type I interferon response within single cells

West Nile virus-inclusive single-cell RNA sequencing reveals heterogeneity in the type I interferon response within single cells

Slow delivery immunization enhances HIV neutralizing antibody and germinal center responses via modulation of immunodominance

Longevity, clonal relationship and transcriptional program of celiac disease-specific plasma cells

Contact Info

Product

Resources

About