Optimization of capture protocols across species targeting up to 32000 genes and their extension to pooled DNA

In-solution based capture is becoming a method of choice for sequencing targeted sequence. We assessed and optimized a capture protocol in 20 different species from 6 different plant genus using kits from 20,000 to 200,000 baits targeting from 300 to 32,000 genes. We evaluated both the effectiveness of the capture protocol and the fold enrichment in targeted sequences. We proposed a protocol with multiplexing up to 96 samples in a single hybridization and showed it was an efficient and cost-effective strategy. We also extended the use of capture to pools of 100 samples and proved the efficiency of the method to assess allele frequency. Using a set of various organisms with different genome sizes, we demonstrated a correlation between the percentage of on-target reads vs. the relative size of the targeted sequences. Altogether, we proposed methods, strategies, cost-efficient protocols and statistics to better evaluate and more effectively use hybridization capture.

A public antibody class recognizes a novel S2 epitope exposed on open conformations of SARS-CoV-2 spike

Delineating the origins and properties of antibodies elicited by SARS-CoV-2 infection and vaccination is critical for understanding their benefits and potential shortcomings. Therefore, we investigated the SARS-CoV-2 spike (S)-reactive B cell repertoire in unexposed individuals by flow cytometry and single-cell sequencing. We found that ∼82% of SARS-CoV-2 S-reactive B cells show a naive phenotype, which represents an unusually high fraction of total human naive B cells (∼0.1%). Approximately 10% of these naive S-reactive B cells shared an IGHV1-69/IGKV3-11 B cell receptor pairing, an enrichment of 18-fold compared to the complete naive repertoire. A proportion of memory B cells, comprising switched (∼0.05%) and unswitched B cells (∼0.04%), was also reactive with S and some of these cells were reactive with ADAMTS13, which is associated with thrombotic thrombocytopenia. Following SARS-CoV-2 infection, we report an average 37-fold enrichment of IGHV1-69/IGKV3-11 B cell receptor pairing in the S-reactive memory B cells compared to the unselected memory repertoire. This class of B cells targets a previously undefined non-neutralizing epitope on the S2 subunit that becomes exposed on S proteins used in approved vaccines when they transition away from the native pre-fusion state because of instability. These findings can help guide the improvement of SARS-CoV-2 vaccines.

Adoptive Transfer Immunization of Mice

This procedure is designed to enrich and expand antibody-forming cells for use in generating monoclonal antibodies. Gamma-irradiation is used to wipe out the immune system in a recipient animal, after which spleen cells that have reverted to memory cells are obtained from syngeneic donor animals and transferred to the irradiated animal, allowing the implanted immune cells to take over. This method can produce an 80-fold enrichment of antibody-producing cells over that obtained in the original immunized animal.

A method to enrich and purify centromeric DNA from human cells

Centromeres are key elements for chromosome segregation. Canonical centromeres are built over long-stretches of tandem repetitive arrays. Despite being quite abundant compared to other loci, centromere sequences overall still represent only 2 to 5% of the human genome, therefore studying their genetic and epigenetic features is a major challenge. Furthermore, sequencing of centromeric regions requires high coverage to fully analyze length and sequence variations, which can be extremely costly. To bypass these issues, we have developed a technique based on selective restriction digestion and size fractionation to enrich for centromeric DNA from human cells. Combining enzymes capable of cutting at high frequency throughout the genome, except within most human centromeres, with size-selection of >20 kb fragments resulted in over 25-fold enrichment in centromeric DNA. Sequencing of the enriched fractions revealed that up to 60% of the enriched material is made of centromeric DNA. This approach has great potential for making sequencing of centromeric DNA more affordable and efficient and for single DNA molecule studies.

Structural variants are a major source of gene expression differences in humans and often affect multiple nearby genes

Structural variants (SVs) are an important source of human genome diversity but their functional effects are not well understood. We mapped 61,668 SVs in 613 individuals with deep genome sequencing data from the GTEx project and measured their effects on gene expression. We estimate that common SVs are causal at 2.66% of eQTLs, which is a 10.5-fold enrichment relative to their abundance in the genome and consistent with prior work using smaller sample sizes. Duplications and deletions were the most impactful variant types, whereas the contribution of mobile element insertions was small (0.12% of eQTLs, 1.9-fold enriched). Multi-tissue analysis of expression effects revealed that gene-altering SVs show significantly more constitutive effects than other variant types, with 62.09% of coding SV-eQTLs active in all tissues with known eQTL activity compared to 23.08% of coding SNV- and indel-eQTLs, while noncoding SVs, SNVs and indels show broadly similar patterns. We also identified 539 rare SVs associated with nearby gene expression outliers. Of these, 62.34% are noncoding SVs that show strong effects on gene expression yet modest enrichment at known regulatory elements, demonstrating that rare noncoding SVs are a major source of gene expression differences but remain difficult to predict from current annotations. Both common and rare noncoding SVs often show strong regional effects on the expression of multiple genes: SV-eQTLs affect an average of 1.82 nearby genes compared to 1.09 genes affected by SNV- and indel-eQTLs, and 21.34% of rare expression-altering SVs show strong effects on 2-9 different genes. We also observe significant effects on rare gene expression changes extending 1 Mb from the SV. This provides a mechanism by which individual noncoding SVs may have strong or pleiotropic effects on phenotypic variation and disease.

Syndecan-2 expression enriches for hematopoietic stem cells and regulates stem cell repopulating capacity

The discovery of novel hematopoietic stem cell (HSC) surface markers can enhance understanding of HSC identity and function. We have discovered a population of primitive bone marrow (BM) HSCs distinguished by their expression of the heparan sulfate proteoglycan, Syndecan-2, which serves as both a marker and regulator of HSC function. Syndecan-2 expression was increased 10-fold in CD150+CD48-CD34-c-Kit+Sca-1+Lineage- cells (long-term - HSCs, LT-HSCs) compared to differentiated hematopoietic cells. Isolation of BM cells based solely on Syndecan-2 surface expression produced a 24-fold enrichment for LT-HSCs, 6-fold enrichment for alpha-catulin+c-kit+ HSCs, and yielded HSCs with superior in vivo repopulating capacity compared to CD150+ cells. Competitive repopulation assays revealed the HSC frequency to be 17-fold higher in Syndecan-2+CD34-KSL cells compared to Syndecan-2-CD34-KSL cells and indistinguishable from CD150+CD34-KSL cells. Syndecan-2 expression also identified nearly all repopulating HSCs within the CD150+CD34-KSL population. Mechanistically, Syndecan-2 regulates HSC repopulating capacity through control of expression of Cdkn1c (p57) and HSC quiescence. Loss of Syndecan-2 expression caused increased HSC cell cycle entry, downregulation of Cdkn1c and loss of HSC long-term - repopulating capacity. Syndecan-2 is a novel marker of HSCs which regulates HSC repopulating capacity via control of HSC quiescence.

Crustal-scale transport of nanoparticle emulsion forms ore deposits

A 10- to 10 000-fold enrichment is required to form economic metal deposits. Such enrichment is achieved through the accumulation of metals transported in hydrothermal fluids from their source to the deposit. The contribution of gold nanoparticle suspensions in fluids is required to form bonanza gold grades. However, as the source of gold is spatially disconnected from the deposit location, it is not known how the transport of gold nanoparticles is achieved. Here we show that metal nanoparticles (Au, AgO, AuAg Cu) are stabilised by colloidal silica in nanoparticle emulsion and transported with the aid of low-density carbonic phases. We document systematic occurrence of metals nanoparticle in five deposits that show a previously unrecognized association with amorphous silica and carbon. Our results demonstrate that stabilisation of metal nanoparticles may be achieved over kilometres through the Earth’s upper crust and offers a step change in our understanding of metalliferous deposit formation.

O-115 Parental whole-exome sequencing allows the discovery of genetic causes of extreme IVF phenotypes such as oocyte/embryo developmental arrest and recurrent low fertilization

Study question Do whole-exome sequencing (WES) data from infertile women provide valuable information for the discovery of genes/pathways involved in extreme IVF phenotypes, i.e. oocyte/embryo developmental arrest? Summary answer The development of a specific bioinformatic WES pipeline revealed known and new candidate genes/pathways for isolated oocyte/embryo developmental failure,providing the foundation to scale up research. What is known already The use of IVF has made it possible to identify extreme and isolated infertility phenotypes such as recurrent low oocytes maturity (LMR), recurrent low fertilization rate (LFR), or preimplantation developmental arrest (PDA) that would remain concealed in natural conception attempts. Recent applications of WES in families with such extreme adverse IVF phenotypes have led to the discovery of new genes and pathways affecting unique functions of gametes and exclusive mechanisms necessary for early embryo development. Here, we apply a tailored bioinformatic approach to WES from women displaying extreme IVF phenotypes to discover new causative genes/pathways involved in unexplained infertility. Study design, size, duration Twenty-two infertile consanguineous women(December 2018-September 2020) suffering from long-term unexplained infertility. Eight cases were classified as PDA (<20% normally developed embryos in > 2 IVF cycles), 8 as LMR (<20% mature oocytes in > 2 IVF cycles), 4 as LFR (<20% of normally fertilized oocytes in > 2 IVF cycles). Two women with recurrent IVF failure (>10 IVF cycles) were also included. A control set of 1660 WES from oocyte donors was used to control for false-positive discoveries. Participants/materials, setting, methods WES at 30X was performed on enrolled women’s gDNA using Illumina short-reads technology. Following annotation, variants were filtered to prioritize putative detrimental variants in genes relevant for oocyte/embryonic development using a previously developed and validated pipeline that minimizes false-positive discoveries. Runs of homozygosity (ROH) within each sample were identified using Refined IBD software. Individual-level single-cell RNAseq (scRNAseq) dataset from 18 human oocytes was used to verify the expression of the identified target genes. Main results and the role of chance The variant prioritization pipeline employed identified 1,160 unique variants in 1,017 genes (average per sample 59.9 sd 8.5). 453 variants were private to this study compared to the 1000 Genomes and gnomAD databases, 3% affecting splicing and/or the gene product length. Significant 5-fold enrichment of 41 genes involved in DNA-damage and repair pathways commonly associated with ovarian function/oocyte quality was observed (p < 0.001). TP53/AKT pathway also showed significant 5-fold enrichment for 45 genes (p < 0.001). This finding is consistent with the known relationship between infertility and cell-cycle/cancer genes. Overall, 66.4% (675/1,017; 95%CI:63.4-69.3) of the targeted genes were expressed in MII human oocytes. Two women (9%) were homozygous carriers of missense pathogenic variants in known candidate genes previously associated to oocyte/embryo developmental arrest (TRIP13, chr5_901344_C/T, CADD percentile 0.999; PADI6, chr1_17394384_C/G, CADD percentile 0.999). Remarkably, four additional women were carriers of high-impact variants in JAKMIP1, a member of a recently characterized family of proteins involved in various cellular processes, including cytoskeleton rearrangement, cell polarization, and intracellular transport. High-impact JAKMP1 variants were never observed in the oocyte donor control dataset. JAKMIP1 mRNA was detected in each individual biological replicate of scRNAseq analysis of MII oocytes with a mean of 6 transcripts per million. Limitations, reasons for caution Functional analysis is ongoing to validate the newly identified genes, data need to be verified in different ethnicities. Nevertheless, this study demonstrates the establishment of a specific and scalable analytical framework that can be employed for the identification of genetic causes in unexplained infertility cases characterized by defective developmental patterns. Wider implications of the findings Scaling up this investigative approach would provide an effective strategy for discovering new genes/pathways in what is considered idiopathic infertility, further defining precision reproductive medicine interventions. Importantly, this study revealed lesions in genetic patterns involved in chronic diseases providing a molecular footprint of the well-established link between infertility and comorbidities. Trial registration number none

A method for the temperature-controlled extraction of DNA from ancient bones

Contamination with microbial and other exogenous DNA poses a significant challenge in the generation of genome-wide sequence data from ancient skeletal remains. Here we describe a method for separating ancient DNA into multiple fractions during DNA extraction by sequential temperature-controlled release of DNA into sodium phosphate buffer. An evaluation of the effectiveness of the method using a set of three ancient bones resulted in between 1.6- and 32-fold enrichment of endogenous DNA compared with regular DNA extraction. For two bones, the method outperformed previous methods of decontaminating ancient bones, including hypochlorite treatment, which resulted in near-complete destruction of DNA in the worst-preserved sample. This extraction method expands the spectrum of methods available for depleting contaminant DNA from ancient skeletal remains.

Prioritization of candidate causal genes for asthma in susceptibility loci derived from UK Biobank

To identify candidate causal genes of asthma, we performed a genome-wide association study (GWAS) in UK Biobank on a broad asthma definition (n = 56,167 asthma cases and 352,255 controls). We then carried out functional mapping through transcriptome-wide association studies (TWAS) and Mendelian randomization in lung (n = 1,038) and blood (n = 31,684) tissues. The GWAS reveals 72 asthma-associated loci from 116 independent significant variants (PGWAS < 5.0E-8). The most significant lung TWAS gene on 17q12-q21 is GSDMB (PTWAS = 1.42E-54). Other TWAS genes include TSLP on 5q22, RERE on 1p36, CLEC16A on 16p13, and IL4R on 16p12, which all replicated in GTEx lung (n = 515). We demonstrate that the largest fold enrichment of regulatory and functional annotations among asthma-associated variants is in the blood. We map 485 blood eQTL-regulated genes associated with asthma and 50 of them are causal by Mendelian randomization. Prioritization of druggable genes reveals known (IL4R, TSLP, IL6, TNFSF4) and potentially new therapeutic targets for asthma.