Comparative Analysis of Droplet-Based Ultra-High-Throughput Single-Cell RNA-Seq Systems.

Since its establishment in 2009, single-cell RNA sequencing (RNA-seq) has been a major driver behind progress in biomedical research. In developmental biology and stem cell studies, the ability to profile single cells confers particular benefits. Although most studies still focus on individual tissues or organs, the recent development of ultra-high-throughput single-cell RNA-seq has demonstrated potential power in characterizing more complex systems or even the entire body. However, although multiple ultra-high-throughput single-cell RNA-seq systems have attracted attention, no systematic comparison of these systems has been performed. Here, with the same cell line and bioinformatics pipeline, we developed directly comparable datasets for each of three widely used droplet-based ultra-high-throughput single-cell RNA-seq systems, inDrop, Drop-seq, and 10X Genomics Chromium. Although each system is capable of profiling single-cell transcriptomes, their detailed comparison revealed the distinguishing features and suitable applications for each system.

SARS-CoV2 infection in whole lung primarily targets macrophages that display subset-specific responses.

Deciphering the initial steps of SARS-CoV-2 infection, that influence COVID-19 outcomes, is challenging because animal models do not always reproduce human biological processes and in vitro systems do not recapitulate the histoarchitecture and cellular composition of respiratory tissues. To address this, we developed an innovative ex vivo model of whole human lung infection with SARS-CoV-2, leveraging a lung transplantation technique. Through single-cell RNA-seq, we identified that alveolar and monocyte-derived macrophages (AMs and MoMacs) were initial targets of the virus. Exposure of isolated lung AMs, MoMacs, classical monocytes and non-classical monocytes (ncMos) to SARS-CoV-2 variants revealed that while all subsets responded, MoMacs produced higher levels of inflammatory cytokines than AMs, and ncMos contributed the least. A Wuhan lineage appeared to be more potent than a D614G virus, in a dose-dependent manner. Amidst the ambiguity in the literature regarding the initial SARS-CoV-2 cell target, our study reveals that AMs and MoMacs are dominant primary entry points for the virus, and suggests that their responses may conduct subsequent injury, depending on their abundance, the viral strain and dose. Interfering on virus interaction with lung macrophages should be considered in prophylactic strategies.

Comparison of the Illumina NextSeq 2000 and GeneMind Genolab M sequencing platforms for spatial transcriptomics.

BACKGROUND: The Illumina sequencing systems demonstrate high efficiency and power and remain the most popular platforms. Platforms with similar throughput and quality profiles but lower costs are under intensive development. In this study, we compared two platforms Illumina NextSeq 2000 and GeneMind Genolab M for 10x Genomics Visium spatial transcriptomics. RESULTS: The performed comparison demonstrates that GeneMind Genolab M sequencing platform produces highly consistent with Illumina NextSeq 2000 sequencing results. Both platforms have similar performance in terms of sequencing quality and detection of UMI, spatial barcode, and probe sequence. Raw read mapping and following read counting produced highly comparable results that is confirmed by quality control metrics and strong correlation between expression profiles in the same tissue spots. Downstream analysis including dimension reduction and clustering demonstrated similar results, and differential gene expression analysis predominantly detected the same genes for both platforms. CONCLUSIONS: GeneMind Genolab M instrument is similar to Illumina sequencing efficacy and is suitable for 10x Genomics Visium spatial transcriptomics.

Single cell transcriptome sequencing on the Nanopore platform with ScNapBar.

The current ecosystem of single cell RNA-seq platforms is rapidly expanding, but robust solutions for single cell and single molecule full- length RNA sequencing are virtually absent. A high-throughput solution that covers all aspects is necessary to study the complex life of mRNA on the single cell level. The Nanopore platform offers long read sequencing and can be integrated with the popular single cell sequencing method on the 10x Chromium platform. However, the high error-rate of Nanopore reads poses a challenge in downstream processing (e.g. for cell barcode assignment). We propose a solution to this particular problem by using a hybrid sequencing approach on Nanopore and Illumina platforms. Our software ScNapBar enables cell barcode assignment with high accuracy, especially if sequencing satura- tion is low. ScNapBar uses unique molecular identifier (UMI) or Naive Bayes probabilistic approaches in the barcode assignment, depending on the available Illumina sequencing depth. We have benchmarked the two approaches on simulated and real Nanopore datasets. We further applied ScNapBar to pools of cells with an active or a silenced non-sense mediated RNA decay pathway. Our Nanopore read assignment distinguishes the respective cell populations and reveals characteristic nonsense-mediated mRNA decay events depending on cell status.

spatialLIBD: an R/Bioconductor package to visualize spatially-resolved transcriptomics data.

BACKGROUND: Spatially-resolved transcriptomics has now enabled the quantification of high-throughput and transcriptome-wide gene expression in intact tissue while also retaining the spatial coordinates. Incorporating the precise spatial mapping of gene activity advances our understanding of intact tissue-specific biological processes. In order to interpret these novel spatial data types, interactive visualization tools are necessary. RESULTS: We describe spatialLIBD, an R/Bioconductor package to interactively explore spatially-resolved transcriptomics data generated with the 10x Genomics Visium platform. The package contains functions to interactively access, visualize, and inspect the observed spatial gene expression data and data-driven clusters identified with supervised or unsupervised analyses, either on the user's computer or through a web application. CONCLUSIONS: spatialLIBD is available at https://bioconductor.org/packages/spatialLIBD . It is fully compatible with SpatialExperiment and the Bioconductor ecosystem. Its functionality facilitates analyzing and interactively exploring spatially-resolved data from the Visium platform.

Single-cell RNA-sequencing of Nicotiana attenuata corolla cells reveals the biosynthetic pathway of a floral scent.

High-throughput single-cell RNA sequencing (scRNA-Seq) identifies distinct cell populations based on cell-to-cell heterogeneity in gene expression. By examining the distribution of the density of gene expression profiles, we can observe the metabolic features of each cell population. Here, we employ the scRNA-Seq technique to reveal the entire biosynthetic pathway of a flower volatile. The corolla of the wild tobacco Nicotiana attenuata emits a bouquet of scents that are composed mainly of benzylacetone (BA). Protoplasts from the N. attenuata corolla limbs and throat cups were isolated at three different time points, and the transcript levels of > 16 000 genes were analyzed in 3756 single cells. We performed unsupervised clustering analysis to determine which cell clusters were involved in BA biosynthesis. The biosynthetic pathway of BA was uncovered by analyzing gene co-expression in scRNA-Seq datasets and by silencing candidate genes in the corolla. In conclusion, the high-resolution spatiotemporal atlas of gene expression provided by scRNA-Seq reveals the molecular features underlying cell-type-specific metabolism in a plant.

Choice of pre-processing pipeline influences clustering quality of scRNA-seq datasets.

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) has quickly become one of the most dominant techniques in modern transcriptome assessment. In particular, 10X Genomics' Chromium system, with its high throughput approach, turn key and thorough user guide made this cutting-edge technique accessible to many laboratories using diverse animal models. However, standard pre-processing, including the alignment and cell filtering pipelines might not be ideal for every organism or tissue. Here we applied an alternative strategy, based on the pseudoaligner kallisto, on twenty-two publicly available single cell sequencing datasets from a wide range of tissues of eight organisms and compared the results with the standard 10X Genomics' Cell Ranger pipeline. RESULTS: In most of the tested samples, kallisto produced higher sequencing read alignment rates and total gene detection rates in comparison to Cell Ranger. Although datasets processed with Cell Ranger had higher cell counts, outside of human and mouse datasets, these additional cells were routinely of low quality, containing low gene detection rates. Thorough downstream analysis of one kallisto processed dataset, obtained from the zebrafish pineal gland, revealed clearer clustering, allowing the identification of an additional photoreceptor cell type that previously went undetected. The finding of the new cluster suggests that the photoreceptive pineal gland is essentially a bi-chromatic tissue containing both green and red cone-like photoreceptors and implies that the alignment and pre-processing pipeline can affect the discovery of biologically-relevant cell types. CONCLUSION: While Cell Ranger favors higher cell numbers, using kallisto results in datasets with higher median gene detection per cell. We could demonstrate that cell type identification was not hampered by the lower cell count, but in fact improved as a result of the high gene detection rate and the more stringent filtering. Depending on the acquired dataset, it can be beneficial to favor high quality cells and accept a lower cell count, leading to an improved classification of cell types.

Identification of immune and non-immune cells in regenerating axolotl limbs by single-cell sequencing.

Immune cells are known to be critical for successful limb regeneration in the axolotl (Ambystoma mexicanum), but many details regarding their identity, behavior, and function are yet to be resolved. We isolated peripheral leukocytes from the blood of adult axolotls and then created two samples for single-cell sequencing: 1) peripheral leukocytes (N = 7889) and 2) peripheral leukocytes with presumptive macrophages from the intraperitoneal cavity (N = 4998). Using k-means clustering, we identified 6 cell populations from each sample that presented gene expression patterns indicative of erythrocyte, thrombocyte, neutrophil, B-cell, T-cell, and myeloid cell populations. A seventh, presumptive macrophage cell population was identified uniquely from sample 2. We then isolated cells from amputated axolotl limbs at 1 and 6 days post-amputation (DPA) and performed single cell sequencing (N = 8272 and 9906 cells respectively) to identify immune and non-immune cell populations. Using k-means clustering, we identified 8 cell populations overall, with the majority of cells expressing erythrocyte-specific genes. Even though erythrocytes predominated, we used an unbiased approach to identify infiltrating neutrophil, macrophage, and lymphocyte populations at both time points. Additionally, populations expressing genes for epidermal cells, fibroblast-like cells, and endothelial cells were also identified. Consistent with results from previous experimental studies, neutrophils were more abundant at 1 DPA than 6 DPA, while macrophages and non-immune cells exhibited inverse abundance patterns. Of note, we identified a small population of fibroblast-like cells at 1 DPA that was represented by considerably more cells at 6 DPA. We hypothesize that these are early progenitor cells that give rise to the blastema. The enriched gene sets from our work will aid future single-cell investigations of immune cell diversity and function during axolotl limb regeneration.

Draft de novo Genome Assembly of the Elusive Jaguarundi, Puma yagouaroundi.

The Puma lineage within the family Felidae consists of 3 species that last shared a common ancestor around 4.9 million years ago. Whole-genome sequences of 2 species from the lineage were previously reported: the cheetah (Acinonyx jubatus) and the mountain lion (Puma concolor). The present report describes a whole-genome assembly of the remaining species, the jaguarundi (Puma yagouaroundi). We sequenced the genome of a male jaguarundi with 10X Genomics linked reads and assembled the whole-genome sequence. The assembled genome contains a series of scaffolds that reach the length of chromosome arms and is similar in scaffold contiguity to the genome assemblies of cheetah and puma, with a contig N50 = 100.2 kbp and a scaffold N50 = 49.27 Mbp. We assessed the assembled sequence of the jaguarundi genome using BUSCO, aligned reads of the sequenced individual and another published female jaguarundi to the assembled genome, annotated protein-coding genes, repeats, genomic variants and their effects with respect to the protein-coding genes, and analyzed differences of the 2 jaguarundis from the reference mitochondrial genome. The jaguarundi genome assembly and its annotation were compared in quality, variants, and features to the previously reported genome assemblies of puma and cheetah. Computational analyzes used in the study were implemented in transparent and reproducible way to allow their further reuse and modification.

Long live the king: chromosome-level assembly of the lion (Panthera leo) using linked-read, Hi-C, and long-read data.

BACKGROUND: The lion (Panthera leo) is one of the most popular and iconic feline species on the planet, yet in spite of its popularity, the last century has seen massive declines for lion populations worldwide. Genomic resources for endangered species represent an important way forward for the field of conservation, enabling high-resolution studies of demography, disease, and population dynamics. Here, we present a chromosome-level assembly from a captive African lion from the Exotic Feline Rescue Center (Center Point, IN) as a resource for current and subsequent genetic work of the sole social species of the Panthera clade. RESULTS: Our assembly is composed of 10x Genomics Chromium data, Dovetail Hi-C, and Oxford Nanopore long-read data. Synteny is highly conserved between the lion, other Panthera genomes, and the domestic cat. We find variability in the length of runs of homozygosity across lion genomes, indicating contrasting histories of recent and possibly intense inbreeding and bottleneck events. Demographic analyses reveal similar ancient histories across all individuals during the Pleistocene except the Asiatic lion, which shows a more rapid decline in population size. We show a substantial influence on the reference genome choice in the inference of demographic history and heterozygosity. CONCLUSIONS: We demonstrate that the choice of reference genome is important when comparing heterozygosity estimates across species and those inferred from different references should not be compared to each other. In addition, estimates of heterozygosity or the amount or length of runs of homozygosity should not be taken as reflective of a species, as these can differ substantially among individuals. This high-quality genome will greatly aid in the continuing research and conservation efforts for the lion, which is rapidly moving towards becoming a species in danger of extinction.

The first complete mitochondrial genome of the sand dollar Sinaechinocyamus mai (Echinoidea: Clypeasteroida).

Morphologic and molecular data often lead to different hypotheses of phylogenetic relationships. Such incongruence has been found in the echinoderm class Echinoidea. In particular, the phylogenetic status of the order Clypeasteroida is not well resolved. Complete mitochondrial genomes are currently available for 29 echinoid species, but no clypeasteroid had been sequenced to date. DNA extracted from a single live individual of Sinaechinocyamus mai was sequenced with 10× Genomics technology. This first complete mitochondrial genome (mitogenome) for the order Clypeasteroida is 15,756 base pairs in length. Phylogenomic analysis based on 34 ingroup taxa belonging to nine orders of the class Echinoidea show congruence between our new genetic inference and published trees based on morphologic characters, but also includes some intriguing differences that imply the need for additional investigation.

Hap10: reconstructing accurate and long polyploid haplotypes using linked reads.

BACKGROUND: Haplotype information is essential for many genetic and genomic analyses, including genotype-phenotype associations in human, animals and plants. Haplotype assembly is a method for reconstructing haplotypes from DNA sequencing reads. By the advent of new sequencing technologies, new algorithms are needed to ensure long and accurate haplotypes. While a few linked-read haplotype assembly algorithms are available for diploid genomes, to the best of our knowledge, no algorithms have yet been proposed for polyploids specifically exploiting linked reads. RESULTS: The first haplotyping algorithm designed for linked reads generated from a polyploid genome is presented, built on a typical short-read haplotyping method, SDhaP. Using the input aligned reads and called variants, the haplotype-relevant information is extracted. Next, reads with the same barcodes are combined to produce molecule-specific fragments. Then, these fragments are clustered into strongly connected components which are then used as input of a haplotype assembly core in order to estimate accurate and long haplotypes. CONCLUSIONS: Hap10 is a novel algorithm for haplotype assembly of polyploid genomes using linked reads. The performance of the algorithms is evaluated in a number of simulation scenarios and its applicability is demonstrated on a real dataset of sweet potato.

The Comparison of Two Single-cell Sequencing Platforms: BD Rhapsody and 10x Genomics Chromium.

The cell is the unit of life for all organisms, and all cells are certainly not the same. So the technology to generate transcription expression or genomic DNA profiles from single cells is crucial. Since its establishment in 2009, single-cell RNA sequencing (scRNA-seq) has emerged as a major driver of progress in biomedical research. During the last three years, several new single-cell sequencing platforms have emerged. Yet there are only a few systematic comparisons of the advantages and limitations of these commonly used platforms. Here we compare two single-cell sequencing platforms: BD Rhapsody and 10x Genomics Chromium, including their different mechanisms and some scRNA-seq results obtained with them.

Assembly and annotation of a draft genome sequence for Glycine latifolia, a perennial wild relative of soybean.

Glycine latifolia (Benth.) Newell & Hymowitz (2n = 40), one of the 27 wild perennial relatives of soybean, possesses genetic diversity and agronomically favorable traits that are lacking in soybean. Here, we report the 939-Mb draft genome assembly of G. latifolia (PI 559298) using exclusively linked-reads sequenced from a single Chromium library. We organized scaffolds into 20 chromosome-scale pseudomolecules utilizing two genetic maps and the Glycine max (L.) Merr. genome sequence. High copy numbers of putative 91-bp centromere-specific tandem repeats were observed in consecutive blocks within predicted pericentromeric regions on several pseudomolecules. No 92-bp putative centromeric repeats, which are abundant in G. max, were detected in G. latifolia or Glycine tomentella. Annotation of the assembled genome and subsequent filtering yielded a high confidence gene set of 54 475 protein-coding loci. In comparative analysis with five legume species, genes related to defense responses were significantly overrepresented in Glycine-specific orthologous gene families. A total of 304 putative nucleotide-binding site (NBS)-leucine-rich-repeat (LRR) genes were identified in this genome assembly. Different from other legume species, we observed a scarcity of TIR-NBS-LRR genes in G. latifolia. The G. latifolia genome was also predicted to contain genes encoding 367 LRR-receptor-like kinases, a family of proteins involved in basal defense responses and responses to abiotic stress. The genome sequence and annotation of G. latifolia provides a valuable source of alternative alleles and novel genes to facilitate soybean improvement. This study also highlights the efficacy and cost-effectiveness of the application of Chromium linked-reads in diploid plant genome de novo assembly.

Tigmint: correcting assembly errors using linked reads from large molecules.

BACKGROUND: Genome sequencing yields the sequence of many short snippets of DNA (reads) from a genome. Genome assembly attempts to reconstruct the original genome from which these reads were derived. This task is difficult due to gaps and errors in the sequencing data, repetitive sequence in the underlying genome, and heterozygosity. As a result, assembly errors are common. In the absence of a reference genome, these misassemblies may be identified by comparing the sequencing data to the assembly and looking for discrepancies between the two. Once identified, these misassemblies may be corrected, improving the quality of the assembled sequence. Although tools exist to identify and correct misassemblies using Illumina paired-end and mate-pair sequencing, no such tool yet exists that makes use of the long distance information of the large molecules provided by linked reads, such as those offered by the 10x Genomics Chromium platform. We have developed the tool Tigmint to address this gap. RESULTS: To demonstrate the effectiveness of Tigmint, we applied it to assemblies of a human genome using short reads assembled with ABySS 2.0 and other assemblers. Tigmint reduced the number of misassemblies identified by QUAST in the ABySS assembly by 216 (27%). While scaffolding with ARCS alone more than doubled the scaffold NGA50 of the assembly from 3 to 8 Mbp, the combination of Tigmint and ARCS improved the scaffold NGA50 of the assembly over five-fold to 16.4 Mbp. This notable improvement in contiguity highlights the utility of assembly correction in refining assemblies. We demonstrate the utility of Tigmint in correcting the assemblies of multiple tools, as well as in using Chromium reads to correct and scaffold assemblies of long single-molecule sequencing. CONCLUSIONS: Scaffolding an assembly that has been corrected with Tigmint yields a final assembly that is both more correct and substantially more contiguous than an assembly that has not been corrected. Using single-molecule sequencing in combination with linked reads enables a genome sequence assembly that achieves both a high sequence contiguity as well as high scaffold contiguity, a feat not currently achievable with either technology alone.

LRTK: a platform agnostic toolkit for linked-read analysis of both human genome and metagenome.

BACKGROUND: Linked-read sequencing technologies generate high-base quality short reads that contain extrapolative information on long-range DNA connectedness. These advantages of linked-read technologies are well known and have been demonstrated in many human genomic and metagenomic studies. However, existing linked-read analysis pipelines (e.g., Long Ranger) were primarily developed to process sequencing data from the human genome and are not suited for analyzing metagenomic sequencing data. Moreover, linked-read analysis pipelines are typically limited to 1 specific sequencing platform. FINDINGS: To address these limitations, we present the Linked-Read ToolKit (LRTK), a unified and versatile toolkit for platform agnostic processing of linked-read sequencing data from both human genome and metagenome. LRTK provides functions to perform linked-read simulation, barcode sequencing error correction, barcode-aware read alignment and metagenome assembly, reconstruction of long DNA fragments, taxonomic classification and quantification, and barcode-assisted genomic variant calling and phasing. LRTK has the ability to process multiple samples automatically and provides users with the option to generate reproducible reports during processing of raw sequencing data and at multiple checkpoints throughout downstream analysis. We applied LRTK on linked reads from simulation, mock community, and real datasets for both human genome and metagenome. We showcased LRTK's ability to generate comparative performance results from preceding benchmark studies and to report these results in publication-ready HTML document plots. CONCLUSIONS: LRTK provides comprehensive and flexible modules along with an easy-to-use Python-based workflow for processing linked-read sequencing datasets, thereby filling the current gap in the field caused by platform-centric genome-specific linked-read data analysis tools.

Sequencing: 10X Genomics 3' HT Assay for Gene Expression.

Single-cell RNA sequencing supports the isolation of individual cells and barcoding of cDNA, specific to each cell of origin. Subsequent sequencing of the generated library yields both the gene expression sequences and the cellular barcode, allowing distinction of gene expression patterns across individual cells. The 10X Genomics 3' HT assay uses a droplet-based method to isolate individual cells within oil emulsions, combined with a gel bead coated in uniquely barcoded primers, specific to each bead. The high-throughput, HT, assay is similar to its predecessor (3' v3.1) in reaction chemistry but utilizes (a) higher numbers of cellular barcodes, (b) a new, proprietary chip designed to target up to 60,000 cells per lane, and (c) captures up to 16 samples per run. The 3' HT assay supports whole cells and nuclei as input, with an approximate 60% capture rate. Here we describe the methods for sample quality control (QC) assays, loading and operation of the Chromium X instrument for cell capture, and cDNA synthesis and library preparation for downstream Illumina sequencing.

Cellular and molecular mechanisms of highly active mesenchymal stem cells in the treatment of senescence of rhesus monkey ovary.

BACKGROUND: Recent studies have shown that umbilical cord mesenchymal stem cells have an anti-aging effect in ovaries, but the cellular and molecular mechanisms of HA-MSC ovarian anti-aging remain to be studied. Therefore, we conducted a 10X Genomics single-nucleus transcriptome sequencing experiment on the ovaries of macaque monkeys after HA-MSC treatment. METHODS: The results of cell subgroup classification were visualized by 10X Genomics single nuclear transcriptome sequencing. The aging model of hGCs was established, and the migration ability of the cells was determined after coculture of HA-MSCs and aging hGCs. The genes screened by single nuclear transcriptional sequencing were verified in vitro by qPCR. RESULTS: Compared with the aging model group, the number of cell receptor pairs in each subgroup of the HA-MSC-treated group increased overall. Treatment with 200 µmol/L H2O2 for 48 h was used as the optimum condition for the induction of hGC senescence. After coculture of noncontact HA-MSCs with senescent hGCs, it was found that HA-MSCs can reverse the cell structure, proliferation ability, senescence condition, expression level of senescence-related genes, and expression level of key genes regulating the senescence pathway in normal hGCs. CONCLUSIONS: HA-MSC therapy can improve the tissue structure and secretion function of the ovary through multiple cellular and molecular mechanisms to resist ovarian aging. In vitro validation experiments further supported the results of single-cell sequencing, which provides evidence supporting a new option for stem cell treatment of ovarian senescence.

Heat Inactivation of Nipah Virus for Downstream Single-Cell RNA Sequencing Does Not Interfere with Sample Quality.

Single-cell RNA sequencing (scRNA-seq) technologies are instrumental to improving our understanding of virus-host interactions in cell culture infection studies and complex biological systems because they allow separating the transcriptional signatures of infected versus non-infected bystander cells. A drawback of using biosafety level (BSL) 4 pathogens is that protocols are typically developed without consideration of virus inactivation during the procedure. To ensure complete inactivation of virus-containing samples for downstream analyses, an adaptation of the workflow is needed. Focusing on a commercially available microfluidic partitioning scRNA-seq platform to prepare samples for scRNA-seq, we tested various chemical and physical components of the platform for their ability to inactivate Nipah virus (NiV), a BSL-4 pathogen that belongs to the group of nonsegmented negative-sense RNA viruses. The only step of the standard protocol that led to NiV inactivation was a 5 min incubation at 85 °C. To comply with the more stringent biosafety requirements for BSL-4-derived samples, we included an additional heat step after cDNA synthesis. This step alone was sufficient to inactivate NiV-containing samples, adding to the necessary inactivation redundancy. Importantly, the additional heat step did not affect sample quality or downstream scRNA-seq results.

A Customizable Suite of Methods to Sequence and Annotate Cattle Antibodies.

Studying the antibody response to infection or vaccination is essential for developing more effective vaccines and therapeutics. Advances in high-throughput antibody sequencing technologies and immunoinformatic tools now allow the fast and comprehensive analysis of antibody repertoires at high resolution in any species. Here, we detail a flexible and customizable suite of methods from flow cytometry, single cell sorting, heavy and light chain amplification to antibody sequencing in cattle. These methods were used successfully, including adaptation to the 10x Genomics platform, to isolate native heavy-light chain pairs. When combined with the Ig-Sequence Multi-Species Annotation Tool, this suite represents a powerful toolkit for studying the cattle antibody response with high resolution and precision. Using three workflows, we processed 84, 96, and 8313 cattle B cells from which we sequenced 24, 31, and 4756 antibody heavy-light chain pairs, respectively. Each method has strengths and limitations in terms of the throughput, timeline, specialist equipment, and cost that are each discussed. Moreover, the principles outlined here can be applied to study antibody responses in other mammalian species.