Pools Of Clones Research Articles

Selection of Antigen Binders from a Chicken Single-Chain Variable Fragment Library.

Antibody production against an antigen of interest is highly efficient in chickens, and the use of chicken antibody libraries in phage display can result in high-affinity single-chain variable fragments (scFvs) for multiple applications. After library preparation from an animal immunized with the antigen of interest, the next step involves the identification of antigen binders. Here, we describe a process for the screening of a phage display chicken library using a technique called bio-panning. It consists of several rounds of binding scFv-displaying phage to antigens, followed by washing, elution, and reamplification. We also describe the steps for assessing clone pools obtained after bio-panning via an ELISA-based procedure known as "phage ELISA" to identify single clones. Last, we provide the steps for using high-throughput sequencing to analyze the pool of selected clones.

Integration of Yeast Episomal/Integrative Plasmid Causes Genotypic and Phenotypic Diversity and Improved Sesquiterpene Production in Metabolically Engineered Saccharomyces cerevisiae.

The variability in phenotypic outcomes among biological replicates in engineered microbial factories presents a captivating mystery. Establishing the association between phenotypic variability and genetic drivers is important to solve this intricate puzzle. We applied a previously developed auxin-inducible depletion of hexokinase 2 as a metabolic engineering strategy for improved nerolidol production in Saccharomyces cerevisiae, and biological replicates exhibit a dichotomy in nerolidol production of either 3.5 or 2.5 g L-1 nerolidol. Harnessing Oxford Nanopore's long-read genomic sequencing, we reveal a potential genetic cause─the chromosome integration of a 2μ sequence-based yeast episomal plasmid, encoding the expression cassettes for nerolidol synthetic enzymes. This finding was reinforced through chromosome integration revalidation, engineering nerolidol and valencene production strains, and generating a diverse pool of yeast clones, each uniquely fingerprinted by gene copy numbers, plasmid integrations, other genomic rearrangements, protein expression levels, growth rate, and target product productivities. Τhe best clone in two strains produced 3.5 g L-1 nerolidol and ∼0.96 g L-1 valencene. Comparable genotypic and phenotypic variations were also generated through the integration of a yeast integrative plasmid lacking 2μ sequences. Our work shows that multiple factors, including plasmid integration status, subchromosomal location, gene copy number, sesquiterpene synthase expression level, and genome rearrangement, together play a complicated determinant role on the productivities of sesquiterpene product. Integration of yeast episomal/integrative plasmids may be used as a versatile method for increasing the diversity and optimizing the efficiency of yeast cell factories, thereby uncovering metabolic control mechanisms.

Marker-free co-selection for successive rounds of prime editing in human cells

Prime editing enables the introduction of precise point mutations, small insertions, or short deletions without requiring donor DNA templates. However, efficiency remains a key challenge in a broad range of human cell types. In this work, we design a robust co-selection strategy through coediting of the ubiquitous and essential sodium/potassium pump (Na+/K+ ATPase). We readily engineer highly modified pools of cells and clones with homozygous modifications for functional studies with minimal pegRNA optimization. This process reveals that nicking the non-edited strand stimulates multiallelic editing but often generates tandem duplications and large deletions at the target site, an outcome dictated by the relative orientation of the protospacer adjacent motifs. Our approach streamlines the production of cell lines with multiple genetic modifications to create cellular models for biological research and lays the foundation for the development of cell-type specific co-selection strategies.

Matrix Metalloproteinase‐9 Promotes Membrane Protrusive Activity in Ovarian Cancer Cells

Matrix Metalloproteinase‐9 (MMP9) is an extracellular endopeptidase associated with ovarian cancer development and progression. To further understand MMP9 contributions to metastatic behavior of an established ovarian cancer cell line (OVCA433), we utilized CRISPR to generate seven cell clones null for MMP9 expression. Analysis of the pooled clones revealed MMP9‐null cells exhibited identical phenotype and growth compared to parental OVCA 433 cells. Cell adhesion and transwell migration towards type I collagen was also not impacted by MMP9 loss. MMP9‐null cells were significantly delayed, however, in EGF‐driven non‐directional filamentous actin‐based membrane protrusions on a variety of matrix substrates. Further, over or re‐expression of MMP9 in OVCA433 or MMP9‐null cells, respectively, was sufficient to drive both lamellipodial and filopodial membrane protrusions on glass. These results indicate MMP9 directly contributes to ovarian cancer cell protrusive activity.

Single-Cell Clonal Tracking in Allogeneic Hematopoietic Stem Cell Transplantation Reveals Time Dependent and Distinct Functional Patterns in Traceable Donor T Cell Clones

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only curative treatment option for various malignant hematological diseases. The therapeutic effect of alloHSCT is a long-lasting graft-versus-leukemia (GvL) effect of the transferred graft. T cells are important mediators of GvL and the longitudinal tracking of T-cell clones from donor to recipient is of particular interest in the setting of alloHSCT as this might provide further insight into mechanisms leading to survival and expansion of particular clones. In a broader sense, we used the unique setting of alloHSCT to study survival and expansion of mature T-cell clones after transfer into an immune cell depleted and allogeneic patient.We used single-cell RNA sequencing (scRNAseq) to integrate immune subset delineation, clone identification and transcriptome information of about 35500 single T cells in peripheral blood of 14 paired donor-recipient samples in four alloHSCT pairs. Donor samples were collected before and after treatment with Granulocyte-Colony Stimulating Factor (GCSF), and recipient samples were collected on days +90 and +180 post-transplant.Looking at common diversity scores of pooled donor versus pooled recipient time points we observe an expected decrease of TCR diversity after transplantation (median inv. Simpson's 379 in donor vs. 20 in recipient samples, p=0.011, Figure 1A). On single cell level, we observe a substantial decrease of unique T-cell clones after transplantation compared to donor samples, which in return means that certain TCR clones markedly expand, contributing to a skewing of the TCR repertoire in the post-transplant course. The majority of these cells represent CD8 effector memory T cells.Our main interest was a better understanding of traceable and persisting T-cell clones. In a first step, we looked at the overall clonal overlap between time points of the different donor-recipient pairs, using only combined TCR alpha and beta chain information to define specific T-cell clones. We find the highest overlaps of T-cell clones between time points within individuals (e.g., Morisita score 0.91 between preGCSF and postGCSF of donor 16 and Morisita score 0.65 between days +90 and +180 of recipient 16, Figure 1B). Additionally, we demonstrate an inter-individual overlap between donors and their respective recipients in all pairs on single cell level (Figure 1C).Next, we compared the differential gene expression of traceable and non-traceable T cell clones and found that the traceable T cell clones exhibit a distinct transcriptional program, characterized by upregulation of genes related to T cell proliferation and chemotaxis as well as antigen presentation, while housekeeping functions such as translation are downregulated.In order to examine the dynamic changes of the T-cell transcriptome, we looked at the differential gene expression at the consecutive time points of pooled traceable clones in all pairs. This shows an induction of an activation pattern during the donor-recipient transfer and post-transplant phase involving genes related to the cell cycle and graft-versus-host disease (Figure 1D). Phenotype analysis via antibody-derived tags accordingly revealed an upregulation of activation markers in the recipients.To our knowledge, this is the first time that longitudinal inter-individual (donor-to-recipient) overlap of single-cell TCR alpha/beta clones is demonstrated in the setting of alloHSCT revealing time point-dependent and distinct functional patterns in traceable donor T cell clones. [Display omitted] DisclosuresPenack: Neovii: Honoraria; MSD: Honoraria; Incyte: Research Funding; Priothera: Consultancy; Therakos: Honoraria; Gilead: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Takeda: Research Funding; Astellas: Honoraria; Jazz: Honoraria; Omeros: Consultancy; Shionogi: Consultancy. Bullinger: Amgen: Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Hexal: Consultancy; Abbvie: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Astellas: Honoraria; Pfizer: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Bayer: Research Funding; Seattle Genetics: Honoraria; Novartis: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria; Menarini: Consultancy; Gilead: Consultancy; Celgene: Consultancy, Honoraria; Sanofi: Honoraria. Na: Bristol Myers Squibb: Research Funding; Shire/Takeda: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding.

Paroxysmal Nocturnal Hemoglobinuria Results from Initial Clonal PIG-a Mosaicism

The pathogenesis of PNH involves alteration of at least one HSC clone, characterized by a somatic mutation in the PIG-A gene. The resulting phenotype appears to offer a growth advantage over other unaffected HSCs, yet the failure of PIG-A mutant clones to expand in animal models suggests that additional factors are necessary for the development of PNH. The association of PNH with aplastic anemia (AA) is evidence of selection pressure by immune attack, but secondary hits in additional genes may also be clonally acquired to render PNH clones competitive irrespective of immune pressure. With the advent of NGS, clonal architecture analysis has been revolutionized. Yet, despite increased resolution, NGS is hampered by the presence of normal/non-clonal cells at varying frequencies in the sequenced sample, often decreasing the detection threshold and precluding recognition of subclonal mosaicism.To study these issues in PNH, 133 patients with PNH (N=33), AA (N=33), and AA/PNH (N=67) were sequenced using deep targeted NGS covering all exons of the PIG-A gene. All patients had a detectable PNH clone by flow cytometry, with the mean WBC clone size of 0.22% in AA, 27.4% in AA/PNH, and 77.1% in PNH. 223 PIG-A mutations were identified in this cohort; 35% were frameshift mutations, 34% missense, 14% splice site, 13% nonsense, and 3% non-frameshift. In unpurified blood, PIG-A mutations were found in 27/33 (82%) of PNH patients, 36/67 (54%) of AA/PNH, and 2/33 (6%) of AA, with a clear association between PNH clone size and the ability to detect mutations by NGS (P<0.0001). In DNA extracted from cells flow sorted by FLAER negativity, detection rates were 26/29 (90%), with an average of nearly 3 mutations per patient, indicating that mechanisms such as microdeletions may affect as many as 10% of PNH or AA/PNH patients and that clonal mosaicism is common. Our results also show that indeed type III and II PNH cells are derived from 2 distinct clones, each with a distinct mutation.To examine disease pathogenesis, we studied 47 patients longitudinally using both PNH flow and deep sequencing. Serial testing illustrates possible modes of evolution: mutations can fluctuate in the contribution to clonal burden over time with the variant allelic frequency of some mutations increasing while others decrease. Linear regression of the number of mutations vs . time from diagnosis suggests that in AA/PNH (N=29), the number of mutant clones detected increases over time (P=.012) while in classic PNH (N=18) the trend is reversed, as the number of mutations detected decreases (P<.0001) and a dominant clone emerges to supply a majority of blood cells.Traditionally, PNH has been considered a monoclonal disease in which a PIG-A mutant HSC clone can outcompete normal hematopoiesis within the context of permissive conditions. Using deep NGS, our results clearly indicate that while a single clone ultimately may dominate, PNH begins as oligoclonal process with the eventual emergence of the most competitive HSC clone(s) populating hematopoietic space. Initially, an environment that “favors” a state of GPI-anchor deficiency thereby allows multiple clones to evolve. Over time, the most “fit” PNH HSC clones dominate, resulting in monoclonality. In addition, our results confirm the presence of distinct mutations with corresponding phenotype through sequencing of sorted type II and type III PNH cells. Clonal drift may also be observed, likely through a newly acquired advantage of one clone within the available pool of GPI-deficient PIGA mutant clones. Our previous work indicated that acquisition of additional enabling mutations results in a more complex clonal architecture comparable to that seen in MDS, an intrinsic mechanism of the selection process that involves mutations in genes altered in MDS. Additional intrinsic hits also explain patient-specific kinetics of PNH evolution and apparent independence of established PNH from the effect of immunosuppression. Thus, while immune selection may provide initial conditions favorable for oligoclonal PNH evolution, once pressure is relieved, intrinsic factors can lead to PNH monoclonality. Alternative mechanisms could include loss of dominance by exhaustion, senescence, or telomere shortening.In sum, our study clearly demonstrates initial polyclonality early in the pathogenesis of PNH with selection of dominant clones during disease duration. DisclosuresMakishima:Yasuda Medical Foundation: Research Funding.

Matrix Metalloproteinase‐9 Promotes Membrane Protrusive Activity in Ovarian Cancer Cells

Matrix Metalloproteinase‐9 (MMP9) is an extracellular endopeptidase associated with ovarian cancer development and progression. To further understand MMP9 contributions to metastatic behavior of an established ovarian cancer cell line (OVCA433), we utilized CRISPR to generate seven cell clones null for MMP9 expression. Analysis of the pooled clones revealed MMP9‐null cells exhibited identical phenotype and growth compared to parental OVCA 433 cells. Cell adhesion and transwell migration towards type I collagen was also not impacted by MMP9 loss. MMP9‐null cells were significantly delayed, however, in non‐directional filamentous actin‐based membrane protrusions on a variety of matrix substrates in response to Epidermal growth factor stimulation. Further, over or re‐expression of MMP9 in OVCA433 or MMP9‐null cells, respectively, was sufficient to drive both lamellipodial and filopodial membrane protrusions on glass. These results indicate MMP9 directly contributes to ovarian cancer protrusive activity.Support or Funding InformationWartburg Undergraduate Research Grants

Mutations in the miR396 binding site of the growth-regulating factor gene VvGRF4 modulate inflorescence architecture in grapevine.

Bunch rot caused by Botrytis cinerea infections is a notorious problem in grapevine cultivation. To produce high quality fruits, grapevine plants are treated with fungicides, which is cost intensive and harmful to the environment. Conversely, loose cluster bunches show a considerably enhanced physical resilience to bunch diseases. With the aim to identify genetic determinants that modulate the development of bunch architecture, we have compared loose and compact 'Pinot noir' clones. Loose cluster architecture was found to be correlated with increased berry size, elongated rachis and elongated pedicels. Using transcriptome analysis in combination with whole genome sequencing, we have identified a growth-regulating factor gene, VvGRF4, upregulated and harbours heterozygous mutations in the loose cluster clones. At late stages of inflorescence development, the mRNA pools of loose cluster clones contain predominantly mRNAs derived from the mutated alleles, which are resistant to miR396 degradation. Expression of the VvGRF4 gene and its mutated variants in Arabidopsis demonstrates that it promotes pedicel elongation. Taken together, VvGRF4 modulates bunch architecture in grapevine 'Pinot noir' clones. This trait can be introduced into other cultivars using marker-assisted breeding or CRISPR-Cas9 technology. Related growth-regulating factors or other genes of the same pathway may have similar functions.

Abstract P2-06-03: Insulin receptor isoform signaling in breast cancer

Abstract Recent data shows that insulin receptor (IR) and the type I insulin-like growth factor receptor (IGF1R) play important roles in breast cancer cell biology. Targeting only IGF1R has not been successful perhaps due to compensation by IR. IR exists in two isoforms, fetal isoform IR-A is a splice variant of IR which excludes exon 11. The adult/metabolic isoform IR-B is the predominant species expressed in normal tissues, while the fetal form IR-A is more highly expressed in breast cancer. IR-A mRNA in endocrine resistant cells is expressed at levels 24-fold higher than IGF1R expression (Gradishar, et al. Clin Cancer Res 22:301 2016 PMID: 26324738). In addition to its homodimer, IR-A can also dimerize with IGF1R to form a hybrid. Homodimer IR-A responds to IGF-II and insulin, while IGF1R/IR-A hybrid can also respond to IGF-I. Previous studies in our lab showed down regulation of IGFIR increases the sensitivity of breast cancer cells to insulin. To further investigate the roles of IR-A and IR-B in breast cancer biology, we cloned IR-A and IR-B isoforms into pLV-mCherry and pLJM1EGFP lentiviral expression vectors. Then MCF-7L breast cancer cells were infected with IR-A, IR-A vector control, IR-B and IR-B vector control. Both pooled and single clones were studied. Our results showed that both IR-A and IR-B were highly expressed in MCF-7L cells with the introduced IR tagged species present at a higher molecular weight confirmed by immunoprecipitation and immunoblot. The lower migrating species was endogenous IR. IR-A-pool/Clones had strong basal IR tyrosine phosphorylation, while IR-B-pool/clones did not. This basal phosphorylation did not activate downstream signaling as measured by pAKT and pErk1,2. IR-A basal phosphorylation was completely inhibited by BMS754807 (0.3μM), a dual IGFIR/IR tyrosine kinase inhibitor. Total levels of IRS1, IRS2, IGF1R, ERa, AKT and Erk1,2 levels were not changed in the over-expressing cells. Ligands-IGF-I (5nM), IGF-II (10nM) and Insulin (10nM) stimulated IGF1R/IR, IRS, AKT and Erk1,2 phosphorylation in IR-A-pool and high expression clone IR-A-G5. Cells expressing IR-A activated downstream signaling (IRS, AKT and Erk1,2) at 0.1nM insulin, while IR-A vector control cells required 1nM insulin. IR-B-pool overexpressing cells were not more sensitive to insulin compared to parental cells. Insulin significantly increased Erk1,2 phosphorylation in IR-A-G5 cell while IGF-I stimulation was minimal. In contrast, Erk1,2 phosphorylation in parental cells and vector control cells was primarily mediated by IGF-I, not insulin. IR-A overexpressing cells were stimulated in monolayer growth by insulin. These data show that IR-A expression, as seen in endocrine resistant breast cancer cells, sensitizes breast cancers to low concentrations of insulin. Thus, IR-A expression could serve as a target in breast cancer. Citation Format: Zhang X, Chan JY, Pan Y, Dong C, Yee D. Insulin receptor isoform signaling in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-03.

A geometry-based algorithm for cloning real grains 2.0

We introduce a computational algorithm to “clone” the grain morphologies of a sample of real grains that have been digitalized. This cloning algorithm allows us to generate an arbitrary number of cloned grains that satisfy the same distributions of morphological features displayed by their parents and can be included into a numerical Discrete Element Method simulation. This study is carried out in three steps. First, distributions of morphological parameters such as aspect ratio, roundness, principal geometric directions, and spherical radius, called the morphological DNA, are extracted from the parents. Second, the geometric stochastic cloning (GSC) algorithm, relying purely on statistical distributions of the aforementioned parameters, is explained, detailed, and used to generate a pool of clones from its parents’ morphological DNA. Third, morphological DNA is extracted from the pool of clones and compared to the one obtained from a similar pool of parents, and the distribution of volume-surface ratio is used to perform quality control. Then, from these results, the error (mutation) in the GSC process is analyzed and used to discuss the algorithm’s drawbacks, knobs (parameters) tuning, as well as potential improvements.

CRISPR-Cas9-Mediated Mutagenesis: Mind the Gap?

CRISPR-Cas9-Mediated Mutagenesis: Mind the Gap?

Whole-Genome Restriction Mapping by "Subhaploid"-Based RAD Sequencing: An Efficient and Flexible Approach for Physical Mapping and Genome Scaffolding.

Assembly of complex genomes using short reads remains a major challenge, which usually yields highly fragmented assemblies. Generation of ultradense linkage maps is promising for anchoring such assemblies, but traditional linkage mapping methods are hindered by the infrequency and unevenness of meiotic recombination that limit attainable map resolution. Here we develop a sequencing-based “in vitro” linkage mapping approach (called RadMap), where chromosome breakage and segregation are realized by generating hundreds of “subhaploid” fosmid/bacterial-artificial-chromosome clone pools, and by restriction site-associated DNA sequencing of these clone pools to produce an ultradense whole-genome restriction map to facilitate genome scaffolding. A bootstrap-based minimum spanning tree algorithm is developed for grouping and ordering of genome-wide markers and is implemented in a user-friendly, integrated software package (AMMO). We perform extensive analyses to validate the power and accuracy of our approach in the model plant Arabidopsis thaliana and human. We also demonstrate the utility of RadMap for enhancing the contiguity of a variety of whole-genome shotgun assemblies generated using either short Illumina reads (300 bp) or long PacBio reads (6–14 kb), with up to 15-fold improvement of N50 (∼816 kb-3.7 Mb) and high scaffolding accuracy (98.1–98.5%). RadMap outperforms BioNano and Hi-C when input assembly is highly fragmented (contig N50 = 54 kb). RadMap can capture wide-range contiguity information and provide an efficient and flexible tool for high-resolution physical mapping and scaffolding of highly fragmented assemblies.

Distinct T cell clones are deleted during the two waves of thymic negative selection

Abstract T cells are the central mediators of adaptive immunity and provide diverse receptors to recognize pathogens. While this diversity protects the host from a wide variety of pathogens, it comes with the potential for harmful autoreactivity. To circumvent autoimmune disease, thymocytes bearing T cell receptors (TCRs) that bind with high affinity to self-peptide/MHC are removed in a process called negative selection, while cells with a low degree of affinity mature into conventional T cells. Recently, it has been shown that the process of negative selection occurs in two stages during thymocyte development, corresponding to medullary or cortical localization in the thymus. Little is known about the self-antigens that are presented and selected against in these locations, and thereby the corresponding T cell repertoires. We examined the T cell repertoires of these two deletion stages by sequencing thymocyte TCRs from mice with impaired negative selection (Bim−/−). These mice were crossed with transgenic Nur77GFP mice, in which GFP expression correlates with TCR signaling strength. Therefore, the rescued autoreactive clones can be identified in Bim deficient mice based on high GFP expression. The two pools of autoreactive clones – CD4+CD8+ double positive thymocytes (largely cortical and CCR7−) and CD4+ single positive thymocytes (predominantly medullary and CCR7+) – had only partially overlapping TCR sequences. As deletion selectively pruned clones, these observations indicate that thymocytes encounter different self-peptide/MHC molecules in the thymic cortex and thymic medulla. To further test if negative selection is TCR intrinsic, we are creating ‘retrogenic’ mice with auto-reactive TCRs identified from each wave.

A strategy to accelerate protein production from a pool of clones in Chinese hamster ovary cells for toxicology studies.

In the biopharmaceutical industry, a clonally derived cell line is typically used to generate material for investigational new drug (IND)-enabling toxicology studies. The same cell line is then used to generate material for clinical studies. If a pool of clones can be used to produce material for IND-enabling toxicology studies (Pool for Tox (PFT) strategy) during the time a lead clone is being selected for clinical material production, the toxicology studies can be accelerated significantly (approximately 4months at Genentech), leading to a potential acceleration of 4months for the IND submission. We explored the feasibility of the PFT strategy with three antibodies-mAb1, mAb2, and mAb3-at the 2L scale. For each antibody, two lead cell lines were identified that generated material with similar product quality to the material generated from the associated pool. For two antibody molecules, mAb1 and mAb2, the material generated by the lead cell lines from 2L bioreactors was tested in an accelerated stability study and was shown to have stability comparable to the material generated by the associated pool. Additionally, we used this approach for two antibody molecules, mAb4 and mAb5, at Tox and GMP production. The materials from the Tox batch at 400L scale and three GMP batches at 2000L scale have comparable product quality attributes for both molecules. Our results demonstrate the feasibility of using a pool of clonally derived cell lines to generate material of similar product quality and stability for use in IND-enabling toxicology studies as was derived from the final production clone, which enabled significant acceleration of timelines into clinical development. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1449-1455, 2017.

Direct determination of diploid genome sequences.

Determining the genome sequence of an organism is challenging, yet fundamental to understanding its biology. Over the past decade, thousands of human genomes have been sequenced, contributing deeply to biomedical research. In the vast majority of cases, these have been analyzed by aligning sequence reads to a single reference genome, biasing the resulting analyses, and in general, failing to capture sequences novel to a given genome. Some de novo assemblies have been constructed free of reference bias, but nearly all were constructed by merging homologous loci into single “consensus” sequences, generally absent from nature. These assemblies do not correctly represent the diploid biology of an individual. In exactly two cases, true diploid de novo assemblies have been made, at great expense. One was generated using Sanger sequencing, and one using thousands of clone pools. Here, we demonstrate a straightforward and low-cost method for creating true diploid de novo assemblies. We make a single library from ∼1 ng of high molecular weight DNA, using the 10x Genomics microfluidic platform to partition the genome. We applied this technique to seven human samples, generating low-cost HiSeq X data, then assembled these using a new “pushbutton” algorithm, Supernova. Each computation took 2 d on a single server. Each yielded contigs longer than 100 kb, phase blocks longer than 2.5 Mb, and scaffolds longer than 15 Mb. Our method provides a scalable capability for determining the actual diploid genome sequence in a sample, opening the door to new approaches in genomic biology and medicine.

A geometry-based algorithm for cloning real grains

We introduce a computational algorithm to “clone” the grain morphologies of a sample of real grains that have been digitalized. This cloning algorithm allows us to generate an arbitrary number of cloned grains that satisfy the same distributions of morphological features displayed by their parents and can be included into a numerical Discrete Element Method simulation. This study is carried out in three steps. First, distributions of morphological parameters such as aspect ratio, roundness, principal geometric directions, and spherical radius, called the morphological DNA, are extracted from the parents. Second, the geometric stochastic cloning (GSC) algorithm, relying purely on statistical distributions of the aforementioned parameters, is explained, detailed, and used to generate a pool of clones from its parents’ morphological DNA. Third, morphological DNA is extracted from the pool of clones and compared to the one obtained from a similar pool of parents, and the distribution of volume-surface ratio is used to perform quality control. Then, from these results, the error (mutation) in the GSC process is analyzed and used to discuss the algorithm’s drawbacks, knobs (parameters) tuning, as well as potential improvements.

Bug mapping and fitness testing of chemically synthesized chromosome X.

Debugging a genome sequence is imperative for successfully building a synthetic genome. As part of the effort to build a designer eukaryotic genome, yeast synthetic chromosome X (synX), designed as 707,459 base pairs, was synthesized chemically. SynX exhibited good fitness under a wide variety of conditions. A highly efficient mapping strategy called pooled PCRTag mapping (PoPM), which can be generalized to any watermarked synthetic chromosome, was developed to identify genetic alterations that affect cell fitness ("bugs"). A series of bugs were corrected that included a large region bearing complex amplifications, a growth defect mapping to a recoded sequence in FIP1, and a loxPsym site affecting promoter function of ATP2 PoPM is a powerful tool for synthetic yeast genome debugging and an efficient strategy for phenotype-genotype mapping.

Estimation of long terminal repeat element content in the Helicoverpa zea genome from high-throughput sequencing of bacterial artificial chromosome pools.

The lepidopteran pest insect Helicoverpa zea feeds on cultivated corn and cotton across the Americas where control remains challenging owing to the evolution of resistance to chemical and transgenic insecticidal toxins, yet genomic resources remain scarce for this species. A bacterial artificial chromosome (BAC) library having a mean genomic insert size of 145 ± 20 kbp was created from a laboratory strain of H. zea, which provides ∼12.9-fold coverage of a 362.8 ± 8.8 Mbp (0.37 ± 0.09 pg) flow cytometry estimated haploid genome size. Assembly of Illumina HiSeq 2000 reads generated from 14 pools that encompassed all BAC clones resulted in 165 485 genomic contigs (N50 = 3262 bp; 324.6 Mbp total). Long terminal repeat (LTR) protein coding regions annotated from 181 contigs included 30 Ty1/copia, 78 Ty3/gypsy, and 73 BEL/Pao elements, of which 60 (33.1%) encoded all five functional polyprotein (pol) domains. Approximately 14% of LTR elements are distributed non-randomly across pools of BAC clones.

Rapid high-throughput cloning and stable expression of antibodies in HEK293 cells

Single-cell based amplification of immunoglobulin variable regions is a rapid and powerful technique for cloning antigen-specific monoclonal antibodies (mAbs) for purposes ranging from general laboratory reagents to therapeutic drugs. From the initial screening process involving small quantities of hundreds or thousands of mAbs through in vitro characterization and subsequent in vivo experiments requiring large quantities of only a few, having a robust system for generating mAbs from cloning through stable cell line generation is essential. A protocol was developed to decrease the time, cost, and effort required by traditional cloning and expression methods by eliminating bottlenecks in these processes. Removing the clonal selection steps from the cloning process using a highly efficient ligation-independent protocol and from the stable cell line process by utilizing bicistronic plasmids to generate stable semi-clonal cell pools facilitated an increased throughput of the entire process from plasmid assembly through transient transfections and selection of stable semi-clonal cell pools. Furthermore, the time required by a single individual to clone, express, and select stable cell pools in a high-throughput format was reduced from 4 to 6months to only 4 to 6weeks.

Building two indica rice reference genomes with PacBio long-read and Illumina paired-end sequencing data.

Over the past 30 years, we have performed many fundamental studies on two Oryza sativa subsp. indica varieties, Zhenshan 97 (ZS97) and Minghui 63 (MH63). To improve the resolution of many of these investigations, we generated two reference-quality reference genome assemblies using the most advanced sequencing technologies. Using PacBio SMRT technology, we produced over 108 (ZS97) and 174 (MH63) Gb of raw sequence data from 166 (ZS97) and 209 (MH63) pools of BAC clones, and generated ~97 (ZS97) and ~74 (MH63) Gb of paired-end whole-genome shotgun (WGS) sequence data with Illumina sequencing technology. With these data, we successfully assembled two platinum standard reference genomes that have been publicly released. Here we provide the full sets of raw data used to generate these two reference genome assemblies. These data sets can be used to test new programs for better genome assembly and annotation, aid in the discovery of new insights into genome structure, function, and evolution, and help to provide essential support to biological research in general.