De novo antibody discovery in human blood from full-length single B cell transcriptomics and matching haplotyped-resolved germline assemblies.

Immunoglobulin (IGH, IGK, IGL) loci in the human genome are highly polymorphic regions that encode the building blocks of the light and heavy chain IG proteins that dimerize to form antibodies. The processes of V(D)J recombination and somatic hypermutation in B cells are responsible for creating an enormous reservoir of highly specific antibodies capable of binding a vast array of possible antigens. However, the antibody repertoire is fundamentally limited by the set of variable (V), diversity (D), and joining (J) alleles present in the germline IG loci. To better understand how the germline IG haplotypes contribute to the expressed antibody repertoire, we combined genome sequencing of the germline IG loci with single-cell transcriptome sequencing of B cells from the same donor. Sequencing and assembly of the germline IG loci captured the IGH locus in a single fully-phased contig where the maternal and paternal contributions to the germline V, D, and J repertoire can be fully resolved. The B cells were collected following a measles, mumps, and rubella (MMR) vaccination, resulting in a population of cells that were activated in response to this specific immune challenge. Single-cell, full-length transcriptome sequencing of these B cells resulted in whole transcriptome characterization of each cell, as well as highly-accurate consensus sequences for the somatically rearranged and hypermutated light and heavy chain IG transcripts. A subset of antibodies synthesized based on their consensus heavy and light chain transcript sequences demonstrated binding to measles antigens and neutralization of measles live virus.

Profiling the genetic determinants of chromatin accessibility with scalable single-cell CRISPR screens.

CRISPR screens have been used to connect genetic perturbations with changes in gene expression and phenotypes. Here we describe a CRISPR-based, single-cell combinatorial indexing assay for transposase-accessible chromatin (CRISPR-sciATAC) to link genetic perturbations to genome-wide chromatin accessibility in a large number of cells. In human myelogenous leukemia cells, we apply CRISPR-sciATAC to target 105 chromatin-related genes, generating chromatin accessibility data for ~30,000 single cells. We correlate the loss of specific chromatin remodelers with changes in accessibility globally and at the binding sites of individual transcription factors (TFs). For example, we show that loss of the H3K27 methyltransferase EZH2 increases accessibility at heterochromatic regions involved in embryonic development and triggers expression of genes in the HOXA and HOXD clusters. At a subset of regulatory sites, we also analyze changes in nucleosome spacing following the loss of chromatin remodelers. CRISPR-sciATAC is a high-throughput, single-cell method for studying the effect of genetic perturbations on chromatin in normal and disease states.

Contrasting Rates of LINE-1 Amplification among New World Primates of the Atelidae Family.

LINE-1 (L1) retrotransposons constitute the dominant category of transposons in mammalian genomes. L1 elements are active in the vast majority of mammals, and only a few cases of L1 extinction have been documented. The only possible case of extinction in primates was suggested for South American spider monkeys. However, these previous studies were based on a single species. We revisited this question with a larger phylogenetic sample, covering all 4 genera of Atelidae and 3 species of spider monkeys. We used an enrichment method to clone recently inserted L1 elements and performed an evolutionary analysis of the sequences. We were able to identify young L1 elements in all taxa, suggesting that L1 is probably still active in all Atelidae examined. However, we also detected considerable variations in the proportion of recent elements indicating that the rate of L1 amplification varies among Atelidae by a 3-fold factor. The extent of L1 amplification in Atelidae remains overall lower than in other New World monkeys. Multiple factors can affect the amplification of L1, such as the demography of the host and the control of transposition. These factors are discussed in the context of host life history.

Contrasted patterns of evolution of the LINE-1 retrotransposon in perissodactyls: the history of a LINE-1 extinction.

BACKGROUND: LINE-1 (L1) is the dominant autonomously replicating non-LTR retrotransposon in mammals. Although our knowledge of L1 evolution across the tree of life has considerably improved in recent years, what we know of L1 evolution in mammals is biased and comes mostly from studies in primates (mostly human) and rodents (mostly mouse). It is unclear if patterns of evolution that are shared between those two groups apply to other mammalian orders. Here we performed a detailed study on the evolution of L1 in perissodactyls by making use of the complete genome of the domestic horse and of the white rhinoceros. This mammalian order offers an excellent model to study the extinction of L1 since the rhinoceros is one of the few mammalian species to have lost active L1. RESULTS: We found that multiple L1 lineages, carrying different 5'UTRs, have been simultaneously active during the evolution of perissodactyls. We also found that L1 has continuously amplified and diversified in horse. In rhinoceros, L1 was very prolific early on. Two successful families were simultaneously active until ~20my ago but became extinct suddenly at exactly the same time. CONCLUSIONS: The general pattern of L1 evolution in perissodactyls is very similar to what was previously described in mouse and human, suggesting some commonalities in the way mammalian genomes interact with L1. We confirmed the extinction of L1 in rhinoceros and we discuss several possible mechanisms.

The Evolution of LINE-1 in Vertebrates.

The abundance and diversity of the LINE-1 (L1) retrotransposon differ greatly among vertebrates. Mammalian genomes contain hundreds of thousands L1s that have accumulated since the origin of mammals. A single group of very similar elements is active at a time in mammals, thus a single lineage of active families has evolved in this group. In contrast, non-mammalian genomes (fish, amphibians, reptiles) harbor a large diversity of concurrently transposing families, which are all represented by very small number of recently inserted copies. Why the pattern of diversity and abundance of L1 is so different among vertebrates remains unknown. To address this issue, we performed a detailed analysis of the evolution of active L1 in 14 mammals and in 3 non-mammalian vertebrate model species. We examined the evolution of base composition and codon bias, the general structure, and the evolution of the different domains of L1 (5'UTR, ORF1, ORF2, 3'UTR). L1s differ substantially in length, base composition, and structure among vertebrates. The most variation is found in the 5'UTR, which is longer in amniotes, and in the ORF1, which tend to evolve faster in mammals. The highly divergent L1 families of lizard, frog, and fish share species-specific features suggesting that they are subjected to the same functional constraints imposed by their host. The relative conservation of the 5'UTR and ORF1 in non-mammalian vertebrates suggests that the repression of transposition by the host does not act in a sequence-specific manner and did not result in an arms race, as is observed in mammals.

Revisiting the evolution of mouse LINE-1 in the genomic era.

BACKGROUND: LINE-1 (L1) is the dominant category of transposable elements in placental mammals. L1 has significantly affected the size and structure of all mammalian genomes and understanding the nature of the interactions between L1 and its mammalian host remains a question of crucial importance in comparative genomics. For this reason, much attention has been dedicated to the evolution of L1. Among the most studied elements is the mouse L1 which has been the subject of a number of studies in the 1980s and 1990s. These seminal studies, performed in the pre-genomic era when only a limited number of L1 sequences were available, have significantly improved our understanding of L1 evolution. Yet, no comprehensive study on the evolution of L1 in mouse has been performed since the completion of this genome sequence. RESULTS: Using the Genome Parsing Suite we performed the first evolutionary analysis of mouse L1 over the entire length of the element. This analysis indicates that the mouse L1 has recruited novel 5'UTR sequences more frequently than previously thought and that the simultaneous activity of non-homologous promoters seems to be one of the conditions for the co-existence of multiple L1 families or lineages. In addition the exchange of genetic information between L1 families is not limited to the 5'UTR as evidence of inter-family recombination was observed in ORF1, ORF2, and the 3'UTR. In contrast to the human L1, there was little evidence of rapid amino-acid replacement in the coiled-coil of ORF1, although this region is structurally unstable. We propose that the structural instability of the coiled-coil domain might be adaptive and that structural changes in this region are selectively equivalent to the rapid evolution at the amino-acid level reported in the human lineage. CONCLUSIONS: The pattern of evolution of L1 in mouse shows some similarity with human suggesting that the nature of the interactions between L1 and its host might be similar in these two species. Yet, some notable differences, particularly in the evolution of ORF1, suggest that the molecular mechanisms involved in host-L1 interactions might be different in these two species.