Biology subjects

Garcia, S.

Publications and source records attributed to Garcia, S..

5 recordsLinked to original sources

bioRxiv2018

Molecularly distinct models of zebrafish Myc-induced B cell leukemia

Zebrafish models of T cell acute lymphoblastic leukemia (T-ALL) have been studied for over a decade, but curiously, robust zebrafish B cell ALL (B-ALL) models had not been described. Recently, our laboratories reported two seemingly closely-related models of zebrafish B-ALL. In these genetic lines, the primary difference is expression of either murine or human transgenic c-MYC, each controlled by the zebrafish rag2 promoter. Here, we compare ALL gene expression in both models. Surprisingly, we find that B-ALL arise in different B cell lineages, with ighm+ vs. ighz+ B-ALL driven by murine Myc vs. human MYC, respectively. Moreover, these B-ALL types exhibit signatures of distinct molecular pathways, further unexpected dissimilarity. Thus, despite sharing analogous genetic makeup, the ALL types in each model are markedly different, proving subtle genetic changes can profoundly impact model organism phenotypes. Investigating the mechanistic differences between mouse and human c-MYC in these contexts may reveal key functional aspects governing MYC-driven oncogenesis in human malignancies.

cancer biology↗

bioRxiv2018

Multiobjective Strain Design: A Framework for Modular Cell Engineering

Diversity of cellular metabolism can be harnessed to produce a large space of molecules. However, development of optimal strains with high product titers, rates, and yields required for industrial production is laborious and expensive. To accelerate the strain engineering process, we have recently introduced a modular cell design concept that enables rapid generation of optimal production strains by systematically assembling a modular cell with an exchangeable production module(s) to produce target molecules efficiently. In this study, we formulated the modular cell design concept as a general multiobjective optimization problem with flexible design objectives derived from mass action. We developed algorithms and an associated software package, named ModCell2 to implement the design. We demonstrated that ModCell2 can systematically identify genetic modifications to design modular cells that can couple with a variety of production modules and exhibit a minimal tradeoff among modularity, performance, and robustness. Analysis of the modular cell designs revealed both intuitive and complex metabolic architectures enabling modular production of these molecules. We envision ModCell2 provides a powerful tool to guide modular cell engineering and sheds light on modular design principles of biological systems.

bioengineering↗

bioRxiv2017

Resolving the Full Spectrum of Human Genome Variation using Linked-Reads

Large-scale population based analyses coupled with advances in technology have demonstrated that the human genome is more diverse than originally thought. To date, this diversity has largely been uncovered using short read whole genome sequencing. However, standard short-read approaches, used primarily due to accuracy, throughput and costs, fail to give a complete picture of a genome. They struggle to identify large, balanced structural events, cannot access repetitive regions of the genome and fail to resolve the human genome into its two haplotypes. Here we describe an approach that retains long range information while harnessing the advantages of short reads. Starting from only [~]1ng of DNA, we produce barcoded short read libraries. The use of novel informatic approaches allows for the barcoded short reads to be associated with the long molecules of origin producing a novel datatype known as Linked-Reads. This approach allows for simultaneous detection of small and large variants from a single Linked-Read library. We have previously demonstrated the utility of whole genome Linked-Reads (lrWGS) for performing diploid, de novo assembly of individual genomes (Weisenfeld et al. 2017). In this manuscript, we show the advantages of Linked-Reads over standard short read approaches for reference based analysis. We demonstrate the ability of Linked-Reads to reconstruct megabase scale haplotypes and to recover parts of the genome that are typically inaccessible to short reads, including phenotypically important genes such as STRC, SMN1 and SMN2. We demonstrate the ability of both lrWGS and Linked-Read Whole Exome Sequencing (lrWES) to identify complex structural variations, including balanced events, single exon deletions, and single exon duplications. The data presented here show that Linked-Reads provide a scalable approach for comprehensive genome analysis that is not possible using short reads alone.

genomics↗

bioRxiv2017

Linked-Read sequencing resolves complex structural variants

Large genomic structural variants (>50bp) are important contributors to disease, yet they remain one of the most difficult types of variation to accurately ascertain, in part because they tend to cluster in duplicated and repetitive regions, but also because the various signals for these events can be challenging to detect with short reads. Clinically, aCGH and karyotype remain the most commonly used assays for genome-wide structural variant (SV) detection, though there is clear potential benefit to an NGS-based assay that accurately detects both SVs and single nucleotide variants. Linked-Read sequencing is a relatively simple, fast, and cost-effective method that is applicable to both genome and targeted assays. Linked-Reads are generated by performing haplotype-level dilution of long input DNA molecules into >1 million barcoded partitions, generating barcoded short reads within those partitions, and then performing short read sequencing in bulk. We performed 30x Linked-Read genome sequencing on a set of 23 samples with known balanced or unbalanced SVs. Twenty-seven of the 29 known events were detected and another event was called as a candidate. Sequence downsampling was performed on a subset to determine the lowest sequence depth required to detect variations. Copy-number variants can be called with as little as 1-2x sequencing depth (5-10Gb) while balanced events require on the order of 10x coverage for variant calls to be made, although specific signal is clearly present at 1-2x sequencing depth. In addition to detecting a full spectrum of variant types with a single test, Linked-Read sequencing provides base-level resolution of breakpoints, enabling complete resolution of even the most complex chromosomal rearrangements.

genomics↗

bioRxiv2017

A Prototype for Modular Cell Engineering

When aiming to produce a target chemical at high yield, titer, and productivity, various combinations of genetic parts available to build the target pathway can generate a large number of strains for characterization. This engineering approach will become increasingly laborious and expensive when seeking to develop desirable strains for optimal production of a large space of biochemicals due to extensive screening. Our recent theoretical development of modular cell (MODCELL) design principles can offer a promising solution for rapid generation of optimal strains by coupling a modular cell and exchangeable production modules in a plug-and-play fashion. In this study, we experimentally validated some designed properties of MODCELL by demonstrating: i) a modular (chassis) cell is required to couple with a production module, a heterologous ethanol pathway, as a testbed, ii) degree of coupling between the modular cell and production modules can be modulated to enhance growth and product synthesis, iii) a modular cell can be used as a host to select an optimal pyruvate decarboxylase (PDC) of the ethanol production module and to help identify a hypothetical PDC protein, and iv) adaptive laboratory evolution based on growth selection of the modular cell can enhance growth and product synthesis rates. We envision that the MODCELL design provides a powerful prototype for modular cell engineering to rapidly create optimal strains for synthesis of a large space of biochemicals.

synthetic biology↗