Biology subjects

Crang, N.

Publications and source records attributed to Crang, N..

7 recordsLinked to original sources

bioRxiv2025

Tech Note - Adaptive Sampling: targeted Oxford Nanopore long-read sequencing

Oxford Nanopore Technology (ONT) has developed a long-read sequencing method known as "adaptive" or "Read Until" sequencing. Unlike Illumina sequencing, this ONT read data is available in real-time during the run, rather than only being available when the sequencing run is complete and it focuses on a specified region of interest (ROI) within the sequence data. We investigated how this technique compared to standard sequencing under a variety of conditions and found it consistently gave a multifold increase in coverage of the ROI compared to standard sequencing, though a strong positional effect was observed for ROI efficacy. Adaptive sampling can be used to selectively enrich or deplete sequences from a library in real-time, enhancing coverage of regions of interest without requiring additional library pre-processing

genomics↗

bioRxiv2025

Isoform-Level Analysis of 10x Genomics Single-Cell cDNA Libraries from Cultured K562 Cells Using Long-Read Sequencing

Integration of Oxford Nanopore Technologies (ONT) long-read sequencing with 10x Genomics single-cell cDNA libraries enables novel transcript detection, isoform analysis and captures full-length gene body coverage. The purpose of the study was the comparison of three approaches for sequencing 10x Genomics Chromium Single Cell cDNA libraries using long-read sequencing: single-cell full-length transcript sequencing by sampling (FLT-seq), the cDNA-PCR Sequencing Kit (SQK-PCS111) and the PCR Expansion Kit (EXP-PCA001). Our aim was to evaluate their efficiency in enriching full-length cDNA fragments, identifying barcodes, detecting novel isoforms and mutations, and characterizing transcript coverage profiles.

genomics↗

bioRxiv2024

Fragmentation of genomic DNA using Covaris g-Tubes prior to library preparation for sequencing

Covaris g-TUBEs can be used to fragment DNA to pre-determined sizes based on the relative centrifugal force that they are run at. They are recommended for use while preparing Oxford Nanopore Technology libraries by the manufacturer. However, the volumes and DNA concentration typically used for ONT libraries are outside the range of the example data provided by Covaris. Here, we ran g-TUBEs at three different relative centrifugal forces and determined the effect on DNA fragmentation in the range 0.5 - 4 {micro}g. This dataset can be used to inform the effective fragmentation of DNA for creating Oxford Nanopore libraries of an optimal size.

molecular biology↗

bioRxiv2024

The energy metabolism of Cupriavidus necator in different trophic conditions

The knallgas bacterium Cupriavidus necator is attracting interest for biotechnological applications due to its extremely versatile metabolism. C. necator can use hydrogen or formic acid as an energy source, fixes CO2 via the Calvin-Benson-Bassham (CBB) cycle, and also grows well on various other organic acids and sugars. Its tri-partite genome is notable for its size and manifold duplications of key genes (CBB cycle, hydrogenases, nitrate reductases). Comparatively little is known about which of these (iso-) enzymes and their cofactors are actually utilized for growth on different energy sources. Here, we investigated the energy metabolism of C. necator H16 by growing a barcoded transposon knockout library on various substrates including succinate, fructose, hydrogen (H2/CO2) and formic acid. The fitness contribution of each gene was determined from enrichment or depletion of the corresponding mutants. Fitness analysis revealed that 1) some, but not all, molybdenum cofactor biosynthesis genes were essential for growth on formate and nitrate respiration. 2) Soluble formate dehydrogenase (FDH) was the dominant enzyme for formate oxidation, not membrane-bound FDH. 3) For hydrogenases, both soluble and membrane-bound enzymes were beneficial for lithoautotrophic growth. 4) Of the six terminal respiratory complexes in C. necator H16, only some are utilized and utilization depends on the energy source. 5) Deletion of hydrogenase-related genes boosted heterotrophic growth, and we show that the relief from associated protein cost is responsible for this phenomenon. This study evaluates the contribution of each of C. necators genes to fitness in biotechnologically relevant growth regimes. Our results illustrate the genomic redundancy of this generalist bacterium, and may inspire future strategies for strain engineering. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/582058v2_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1aac357org.highwire.dtl.DTLVardef@1fe970corg.highwire.dtl.DTLVardef@16ea53dorg.highwire.dtl.DTLVardef@122498d_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIa barcoded transposon library was used to assess gene fitness for hydrogenase, formate dehydrogenase and electron transport chain complexes C_LIO_LIthe utilization of terminal respiratory complexes is substrate specific C_LIO_LIsoluble formate dehydrogenase is more important than the membrane-bound enzyme C_LIO_LIsoluble hydrogenase and membrane-bound hydrogenase are equally utilized C_LIO_LIinactivation of hydrogenase and its accessory genes leads to faster heterotrophic growth C_LIO_LIwe demonstrate that faster growth is caused by the relief of protein synthesis burden C_LIO_LIfitness data is available in an interactive app at https://m-jahn.shinyapps.io/ShinyLib/ C_LI

microbiology↗

bioRxiv2022

Thermodynamic limitations of metabolic strategies for PHB production from formate and fructose in Cupriavidus necator

The chemolithotroph Cupriavidus necator H16 is known as a natural producer of the bioplastic-polymer PHB, as well as for its metabolic versatility to utilize different substrates, including formate as the sole carbon and energy source. Depending on the entry point of the substrate, this versatility requires adjustment of the thermodynamic landscape to maintain sufficiently high driving forces for biological processes. Here we employed a model of the core metabolism of C. necator H16 to analyze the thermodynamic driving forces and PHB yields of different metabolic engineering strategies. For this, we enumerated elementary flux modes (EFMs) of the network and evaluated their PHB yields as well as thermodynamics via Max-min driving force (MDF) analysis and random sampling of driving forces. A heterologous ATP:citrate lyase reaction was predicted to increase driving force for producing acetyl-CoA. A heterologous phosphoketolase reaction was predicted to increase maximal PHB yields as well as driving forces. These enzymes were verified experimentally to enhance PHB titers between 60 and 300% in select conditions. The EFM analysis also revealed that metabolic strategies for PHB production from formate may be limited by low driving forces through citrate lyase and aconitase, as well as cofactor balancing, and identified reactions of the core metabolism associated with low and high PHB yield. The findings of this study aid in understanding metabolic adaptation. Furthemore, the outlined approach will be useful in designing metabolic engineering strategies in other non-model bacteria. HighlightsO_LIElementary flux modes of C. necator for PHB synthesis from fructose and formate. C_LIO_LIMetabolite sampling identified common reactions among EFMs with low driving force. C_LIO_LIPHB from formate shows low driving forces for aconitase, citrate lyase, NADPH synthesis. C_LIO_LIPhosphoketolase and ATP citrate lyase increased driving forces and PHB production. C_LI

bioengineering↗

bioRxiv2021

Protein allocation and utilization in the versatile chemolithoautotroph Cupriavidus necator

Bacteria must balance the different needs for substrate assimilation, growth functions, and resilience in order to thrive in their environment. Of all cellular macromolecules, the bacterial proteome is by far the most important resource and its size is limited. Here, we investigated how the highly versatile knallgas bacterium Cupriavidus necator reallocates protein resources when grown on different limiting substrates and with different growth rates. We determined protein quantity by mass spectrometry and estimated enzyme utilization by resource balance analysis modeling. We found that C. necator invests a large fraction of its proteome in functions that are hardly utilized. Of the enzymes that are utilized, many are present in excess abundance. One prominent example is the strong expression of CBB cycle genes such as Rubisco during growth on fructose. Modeling and mutant competition experiments suggest that CO2-reassimilation through Rubisco does not provide a fitness benefit for heterotrophic growth, but is rather an investment in readiness for autotrophy. HighlightsO_LIA large fraction of the C. necator proteome is not utilized and not essential C_LIO_LIHighly utilized enzymes are more abundant and less variable C_LIO_LIAutotrophy related enzymes are largely underutilized C_LIO_LIRe-assimilation of CO2 via the CBB cycle is unlikely to provide a fitness benefit C_LI

systems biology↗

bioRxiv2021

Metabolic constraints on nitrogen fixation by rhizobia in legume nodules

Rhizobia induce nodule formation on legume roots and differentiate into bacteroids, which use plant-derived dicarboxylates as energy and electron sources for reduction of atmospheric N2 into ammonia for secretion to plants. Using heterogeneous genome-scale datasets, we reconstructed a model of bacteroid metabolism to investigate the effects of varying dicarboxylate and oxygen supply on carbon and nitrogen allocation. Modelling and 13C metabolic flux analysis in bacteroids indicate that microaerobiosis restricts the decarboxylating arm of the TCA cycle and limits ammonia assimilation into glutamate. Catabolism of dicarboxylates induces a higher oxygen demand but also a higher NADH/NAD+ ratio compared to sugars. Carbon polymer synthesis and alanine secretion by bacteroids facilitate redox balance in microaerobic nodules with alanine secretion increasing as oxygen tension decreases. Our results provide a framework for understanding fundamental constraints on rhizobial metabolism during symbiotic nitrogen fixation.

microbiology↗