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901.
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903.
High‐throughput sequencing has revolutionized population and conservation genetics. RAD sequencing methods, such as 2b‐RAD, can be used on species lacking a reference genome. However, transferring protocols across taxa can potentially lead to poor results. We tested two different IIB enzymes (AlfI and CspCI) on two species with different genome sizes (the loggerhead turtle Caretta caretta and the sharpsnout seabream Diplodus puntazzo) to build a set of guidelines to improve 2b‐RAD protocols on non‐model organisms while optimising costs. Good results were obtained even with degraded samples, showing the value of 2b‐RAD in studies with poor DNA quality. However, library quality was found to be a critical parameter on the number of reads and loci obtained for genotyping. Resampling analyses with different number of reads per individual showed a trade‐off between number of loci and number of reads per sample. The resulting accumulation curves can be used as a tool to calculate the number of sequences per individual needed to reach a mean depth ≥20 reads to acquire good genotyping results. Finally, we demonstrated that selective‐base ligation does not affect genomic differentiation between individuals, indicating that this technique can be used in species with large genome sizes to adjust the number of loci to the study scope, to reduce sequencing costs and to maintain suitable sequencing depth for a reliable genotyping without compromising the results. Here, we provide a set of guidelines to improve 2b‐RAD protocols on non‐model organisms with different genome sizes, helping decision‐making for a reliable and cost‐effective genotyping.  相似文献   
904.
The high‐throughput capacities of the Illumina sequencing platforms and the possibility to label samples individually have encouraged wide use of sample multiplexing. However, this practice results in read misassignment (usually <1%) across samples sequenced on the same lane. Alarmingly high rates of read misassignment of up to 10% were reported for lllumina sequencing machines with exclusion amplification chemistry. This may make use of these platforms prohibitive, particularly in studies that rely on low‐quantity and low‐quality samples, such as historical and archaeological specimens. Here, we use barcodes, short sequences that are ligated to both ends of the DNA insert, to directly quantify the rate of index hopping in 100‐year old museum‐preserved gorilla (Gorilla beringei) samples. Correcting for multiple sources of noise, we identify on average 0.470% of reads containing a hopped index. We show that sample‐specific quantity of misassigned reads depends on the number of reads that any given sample contributes to the total sequencing pool, so that samples with few sequenced reads receive the greatest proportion of misassigned reads. This particularly affects ancient DNA samples, as these frequently differ in their DNA quantity and endogenous content. Through simulations we show that even low rates of index hopping, as reported here, can lead to biases in ancient DNA studies when multiplexing samples with vastly different quantities of endogenous material.  相似文献   
905.
Dendrolimus spp. are important destructive pests of conifer forests, and Dendrolimus punctatus Walker (Lepidoptera; Lasiocampidae) is the most widely distributed Dendrolimus species. During periodic outbreaks, this species is said to make “fire without smoke” because large areas of pine forest can be quickly and heavily damaged. Yet, little is known about the molecular mechanisms that underlie the unique ecological characteristics of this forest insect. Here, we combined Pacific Biosciences (PacBio) RSII single‐molecule long reads and high‐throughput chromosome conformation capture (Hi‐C) genomics‐linked reads to produce a high‐quality, chromosome‐level reference genome for D. punctatus. The final assembly was 614 Mb with contig and scaffold N50 values of 1.39 and 22.15 Mb, respectively, and 96.96% of the contigs anchored onto 30 chromosomes. Based on the prediction, this genome contained 17,593 protein‐coding genes and 56.16% repetitive sequences. Phylogenetic analyses indicated that D. punctatus diverged from the common ancestor of Hyphantria cunea, Spodoptera litura and Thaumetopoea pityocampa ~ 108.91 million years ago. Many gene families that were expanded in the D. punctatus genome were significantly enriched for the xenobiotic biodegradation system, especially the cytochrome P450 gene family. This high‐quality, chromosome‐level reference genome will be a valuable resource for understanding mechanisms of D. punctatus outbreak and host resistance adaption. Because this is the first Lasiocampidae insect genome to be sequenced, it also will serve as a reference for further comparative genomics.  相似文献   
906.
907.
The shift from straw incorporation to biofuel production entails emissions from production, changes in soil organic carbon (SOC) and through the provision of (co‐)products and entailed displacement effects. This paper analyses changes in greenhouse gas (GHG) emissions arising from the shift from straw incorporation to biomethane and bioethanol production. The biomethane concept comprises comminution, anaerobic digestion and amine washing. It additionally provides an organic fertilizer. Bioethanol production comprises energetic use of lignin, steam explosion, enzymatic hydrolysis and co‐fermentation. Additionally, feed is provided. A detailed consequential GHG balance with in‐depth focus on the time dependency of emissions is conducted: (a) the change in the atmospheric load of emissions arising from the change in the temporal occurrence of emissions comparing two steady states (before the shift and once a new steady state has established); and (b) the annual change in overall emissions over time starting from the shift are assessed. The shift from straw incorporation to biomethane production results in net changes in GHG emissions of (a) ?979 (?436 to ?1,654) and (b) ?955 (?220 to ?1,623) kg CO2‐eq. per tdry matter straw converted to biomethane (minimum and maximum). The shift to bioethanol production results in net changes of (a) ?409 (?107 to ?610) and (b) ?361 (57 to ?603) kg CO2‐eq. per tdry matter straw converted to bioethanol. If the atmospheric load of emissions arising from different timing of emissions is neglected in case (a), the change in GHG emissions differs by up to 54%. Case (b) reveals carbon payback times of 0 (0–49) and 19 (1–100) years in case of biomethane and bioethanol production, respectively. These results demonstrate that the detailed inclusion of temporal aspects into GHG balances is required to get a comprehensive understanding of changes in GHG emissions induced by the introduction of advanced biofuels from agricultural residues.  相似文献   
908.
In vitro 3D tissue‐engineered (TE) structures have been shown to better represent in vivo tissue morphology and biochemical pathways than monolayer culture, and are less ethically questionable than animal models. However, to create systems with even greater relevance, multiple integrated tissue systems should be recreated in vitro. In the present study, the effects and conditions most suitable for the co‐culture of TE skeletal muscle and bone are investigated. High‐glucose Dulbecco's modified Eagle medium (HG‐DMEM) supplemented with 20% fetal bovine serum followed by HG‐DMEM with 2% horse serum is found to enable proliferation of both C2C12 muscle precursor cells and TE85 human osteosarcoma cells, fusion of C2C12s into myotubes, as well as an upregulation of RUNX2/CBFa1 in TE85s. Myotube formation is also evident within indirect contact monolayer cultures. Finally, in 3D co‐cultures, TE85 collagen/hydroxyapatite constructs have significantly greater expression of RUNX2/CBFa1 and osteocalcin/BGLAP in the presence of collagen‐based C2C12 skeletal muscle constructs; however, fusion within these constructs appears reduced. This work demonstrates the first report of the simultaneous co‐culture and differentiation of 3D TE skeletal muscle and bone, and represents a significant step toward a full in vitro 3D musculoskeletal junction model.  相似文献   
909.
Scyllo‐inositol (SI), a stereoisomer of inositol, is regarded as a promising therapeutic agent for Alzheimer's disease. Here, an in vitro cofactor‐balance biotransformation for the production of SI from myo‐inositol (MI) by thermophilic myo‐inositol 2‐dehydrogenase (IDH) and scyllo‐inositol 2‐dehydrogenase (SIDH) is presented. These two enzymes (i.e., IDH and SIDH from Geobacillus kaustophilus) are co‐expressed in Escherichia coli BL21(DE3), and E. coli cells containing the two enzymes are permeabilized by heat treatment as whole‐cell catalysts to convert MI to SI. After condition optimizations about permeabilized temperature, reaction temperature, and initial MI concentration, about 82 g L?1 of SI is produced from 250 g L?1 of MI within 24 h without any cofactor supplementation. This final titer of SI produced is the highest to the authors’ limited knowledge. This study provides a promising method for the large‐scale industrial production of SI.  相似文献   
910.
Photosynthetic generation of reducing power makes cyanobacteria an attractive host for biochemical reduction compared to cell‐free and heterotrophic systems, which require burning of additional resources for the supply of reducing equivalent. Here, using xylitol synthesis as an example, efficient uptake and reduction of xylose photoautotrophically in Synechococcus elongatus PCC7942 are demonstrated upon introduction of an effective xylose transporter from Escherichia coli (Ec‐XylE) and the NADPH‐dependent xylose reductase from Candida boidinii (Cb‐XR). Simultaneous activation of xylose uptake and matching of cofactor specificity enabled an average xylitol yield of 0.9 g g?1 xylose and a maximum productivity of about 0.15 g L?1 day?1 OD?1 with increased level of xylose supply. While long‐term cellular maintenance still appears challenging, high‐density conversion of xylose to xylitol using concentrated resting cell further pushes the titer of xylitol formation to 33 g L?1 in six days with 85% of maximum theoretical yield. While the results show that the unknown dissipation of xylose can be minimized when coupled to a strong reaction outlet, it remains to be the major hurdle hampering the yield despite the reported inability of cyanobacteria to metabolize xylose.  相似文献   
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