State of the Art of Genetic Engineering in Potato: From the First Report to Its Future Potential

Potato (Solanum tuberosum L.) is a crop of world importance that produces tubers of high nutritional quality. It is considered one of the promising crops to overcome the challenges of poverty and hunger worldwide. However, it is exposed to different biotic and abiotic stresses that can cause significant losses in production. Thus, potato is a candidate of special relevance for improvements through conventional breeding and biotechnology. Since conventional breeding is time-consuming and challenging, genetic engineering provides the opportunity to introduce/switch-off genes of interest without altering the allelic combination that characterize successful commercial cultivars or to induce targeted sequence modifications by New Breeding Techniques. There is a variety of methods for potato improvement via genetic transformation. Most of them incorporate genes of interest into the nuclear genome; nevertheless, the development of plastid transformation protocols broadened the available approaches for potato breeding. Although all methods have their advantages and disadvantages, Agrobacterium-mediated transformation is the most used approach. Alternative methods such as particle bombardment, protoplast transfection with polyethylene glycol and microinjection are also effective. Independently of the DNA delivery approach, critical steps for a successful transformation are a rapid and efficient regeneration protocol and a selection system. Several critical factors affect the transformation efficiency: vector type, insert size, Agrobacterium strain, explant type, composition of the subculture media, selective agent, among others. Moreover, transient or stable transformation, constitutive or inducible promoters, antibiotic/herbicide resistance or marker-free strategies can be considered. Although great efforts have been made to optimize all the parameters, potato transformation protocols are highly genotype-dependent. Genome editing technologies provide promising tools in genetic engineering allowing precise modification of targeted sequences. Interestingly, transient expression of genome editing components in potato protoplasts was reported to generate edited plants without the integration of any foreign DNA, which is a valuable aspect from both a scientific and a regulatory perspective. In this review, current challenges and opportunities concerning potato genetic engineering strategies developed to date are discussed. We describe their critical parameters and constrains, and the potential application of the available tools for functional analyses or biotechnological purposes. Public concerns and safety issues are also addressed.

Evaluating whole-genome sequencing quality metrics for enteric pathogen outbreaks

Background Whole genome sequencing (WGS) has gained increasing importance in responses to enteric bacterial outbreaks. Common analysis procedures for WGS, single nucleotide polymorphisms (SNPs) and genome assembly, are highly dependent upon WGS data quality. Methods Raw, unprocessed WGS reads from Escherichia coli, Salmonella enterica, and Shigella sonnei outbreak clusters were characterized for four quality metrics: PHRED score, read length, library insert size, and ambiguous nucleotide composition. PHRED scores were strongly correlated with improved SNPs analysis results in E. coli and S. enterica clusters. Results Assembly quality showed only moderate correlations with PHRED scores and library insert size, and then only for Salmonella. To improve SNP analyses and assemblies, we compared seven read-healing pipelines to improve these four quality metrics and to see how well they improved SNP analysis and genome assembly. The most effective read healing pipelines for SNPs analysis incorporated quality-based trimming, fixed-width trimming, or both. The Lyve-SET SNPs pipeline showed a more marked improvement than the CFSAN SNP Pipeline, but the latter performed better on raw, unhealed reads. For genome assembly, SPAdes enabled significant improvements in healed E. coli reads only, while Skesa yielded no significant improvements on healed reads. Conclusions PHRED scores will continue to be a crucial quality metric albeit not of equal impact across all types of analyses for all enteric bacteria. While trimming-based read healing performed well for SNPs analyses, different read healing approaches are likely needed for genome assembly or other, emerging WGS analysis methodologies.

Imageless robotic-assisted revision arthroplasty from UKA to TKA

Background and objective It is evident from the national joint registries that numbers of revision knee arthroplasty operations are rising. The aim of this article is to introduce a new robotic-assisted approach in UKA to TKA revision arthroplasty and investigate the alignment accuracy, implant component use and surgery time and to compare it to primary robotic-assisted TKA arthroplasty. Methods This retrospective, case-control study included patients undergoing image-less robotic-assisted revision arthroplasty from UKA to TKA (n = 20) and patients undergoing image-less robotic-assisted primary TKA (control group, n = 20) from 11/2018 to 07/2020. The control group was matched based on the BMI and natural alignment. Comparison of groups was based on postoperative alignment, outlier rate, tibial insert size, lateral bone resection depth, incision-to-wound closure time. All surgeries were performed by a single senior surgeon using the same bi-cruciate stabilizing TKA system. Statistical analysis consisted of parametric t‑testing and Fisher’s exact test with a level of significance of p < 0.05. Results The two groups showed no differences in mean BMI, natural alignment (p > 0.05) and mean overall limb alignment. No outlier was found for OLA and slope analysis. The smallest insert size (9 mm) was used in 70% of the cases in the revision group (n = 14) and in 90% of the cases in the primary group (n = 18, p = 0.24), distal femoral and tibial resection depth showed no statistical difference (p > 0.05). The incision to wound closure time was longer in the revision group but showed no significant difference. Conclusion Image-less robotic-assisted revision arthroplasty from UKA to TKA showed a comparable surgery time, and alignment accuracy in comparison to primary robotic-assisted TKA. Comparable bone preservation and subsequent tibial insert size use was observed for both groups.

Non-codon Optimized PiggyBac Transposase Induces Developmental Brain Aberrations: A Call for in vivo Analysis

In this perspective article, we briefly review tools for stable gain-of-function expression to explore key fate determinants in embryonic brain development. As the piggyBac transposon system has the highest insert size, a seamless integration of the transposed sequence into the host genome, and can be delivered by transfection avoiding viral vectors causing an immune response, we explored its use in the murine developing forebrain. The original piggyBac transposase PBase or the mouse codon-optimized version mPB and the construct to insert, contained in the piggyBac transposon, were introduced by in utero electroporation at embryonic day 13 into radial glia, the neural stem cells, in the developing dorsal telencephalon, and analyzed 3 or 5 days later. When using PBase, we observed an increase in basal progenitor cells, often accompanied by folding aberrations. These effects were considerably ameliorated when using the piggyBac plasmid together with mPB. While size and strength of the electroporated region was not correlated to the aberrations, integration was essential and the positive correlation to the insert size implicates the frequency of transposition as a possible mechanism. We discuss this in light of the increase in transposing endogenous viral vectors during mammalian phylogeny and their role in neurogenesis and radial glial cells. Most importantly, we aim to alert the users of this system to the phenotypes caused by non-codon optimized PBase application in vivo.

Numerical Study of Heat Transfer Intensification in a Circular Tube Using a Thin, Radiation-Absorbing Insert. Part 1: Thermo-Hydraulic Characteristics

The presented paper, which is the first of two parts, shows the results of numerical investigations of a heat exchanger channel in the form of a cylindrical tube with a thin insert. The insert, placed concentrically in the pipe, uses the phenomenon of thermal radiation absorption to intensify the heat transfer between the pipe wall and the gas. Eight geometric configurations of the insert size were numerically investigated using CFD software, varying its diameter from 20% to 90% of the pipe diameter and obtaining the thermal-flow characteristics for each case. The tests were conducted for a range of numbers Re = 5000–100,000 and a constant temperature difference between the channel wall and the average gas temperature of ∆T = 100 °C. The results show that the highest increase in the Nu number was observed for the inserts with diameters of 0.3 and 0.4 of the channel diameter, while the highest flow resistance was noted for the inserts with diameters of 0.6–0.7 of the channel diameter. The f/fs(Re) and Nu/Nus(Re) ratios are shown on graphs indicating how much the flow resistance and heat transfer increased compared to the pipe without an insert. Two methods of calculating the Nu number are also presented and analysed. In the first one, the average fluid temperature of the entire pipe volume was used to calculate the Nu number, and in the second, only the average fluid temperature of the annular portion formed by the insert was used. The second one gives much larger Nu/Nus ratio values, reaching up to 8–9 for small Re numbers.

Whole genome resequencing data sets of different species from Pistacia genus

Objectives Pistacia genus belongs to the flowering plants in the cashew family and contains at least 11 species. The whole-genome resequencing data of different species from Pistacia genus are described herein. The data reported here will be useful for better understand the adaptive evolution, demographic history, genetic diversity, population structure, and domestication of pistachio. Data description Genomic DNA was isolated from fresh leaves and used to construct libraries with insert size of 350 bp. Sequence libraries were made and sequenced on the Illumina Hiseq 4000 platform to produce 150 bp paired-end reads. A total number of 4,851,118,730 billion reads (ranging from 33,305,900 to 34,990,618 reads per sample) were created across all samples. We produced a total of 727.67 Gbp data which have been deposited in the Genome Sequence Archive (GSA) database with the Accession of CRA000978. All of the data are also available as the sequence read archive (SRA) format in the National Center for Biotechnology Information (NCBI) with identifier of SRP189222, mirroring our deposited data in GSA.

SAUTE: sequence assembly using target enrichment

Background Illumina is the dominant sequencing technology at this time. Short length, short insert size, some systematic biases, and low-level carryover contamination in Illumina reads continue to make assembly of repeated regions a challenging problem. Some applications also require finding multiple well supported variants for assembled regions. Results To facilitate assembly of repeat regions and to report multiple well supported variants when a user can provide target sequences to assist the assembly, we propose SAUTE and SAUTE_PROT assemblers. Both assemblers use de Bruijn graph on reads. Targets can be transcripts or proteins for RNA-seq reads and transcripts, proteins, or genomic regions for genomic reads. Target sequences are nucleotide and protein sequences for SAUTE and SAUTE_PROT, respectively. Conclusions For RNA-seq, comparisons with Trinity, rnaSPAdes, SPAligner, and SPAdes assembly of reads aligned to target proteins by DIAMOND show that SAUTE_PROT finds more coding sequences that translate to benchmark proteins. Using AMRFinderPlus calls, we find SAUTE has higher sensitivity and precision than SPAdes, plasmidSPAdes, SPAligner, and SPAdes assembly of reads aligned to target regions by HISAT2. It also has better sensitivity than SKESA but worse precision.

SIns: A Novel Insertion Detection Approach Based on Soft-Clipped Reads

As a common type of structural variation, an insertion refers to the addition of a DNA sequence into an individual genome and is usually associated with some inherited diseases. In recent years, many methods have been proposed for detecting insertions. However, the accurate calling of insertions is also a challenging task. In this study, we propose a novel insertion detection approach based on soft-clipped reads, which is called SIns. First, based on the alignments between paired reads and the reference genome, SIns extracts breakpoints from soft-clipped reads and determines insertion locations. The insert size information about paired reads is then further clustered to determine the genotype, and SIns subsequently adopts Minia to assemble the insertion sequences. Experimental results show that SIns can achieve better performance than other methods in terms of the F-score value for simulated and true datasets.

A study of transposable element-associated structural variations (TASVs) using a de novo-assembled Korean genome

Advances in next-generation sequencing (NGS) technology have made personal genome sequencing possible, and indeed, many individual human genomes have now been sequenced. Comparisons of these individual genomes have revealed substantial genomic differences between human populations as well as between individuals from closely related ethnic groups. Transposable elements (TEs) are known to be one of the major sources of these variations and act through various mechanisms, including de novo insertion, insertion-mediated deletion, and TE–TE recombination-mediated deletion. In this study, we carried out de novo whole-genome sequencing of one Korean individual (KPGP9) via multiple insert-size libraries. The de novo whole-genome assembly resulted in 31,305 scaffolds with a scaffold N50 size of 13.23 Mb. Furthermore, through computational data analysis and experimental verification, we revealed that 182 TE-associated structural variation (TASV) insertions and 89 TASV deletions contributed 64,232 bp in sequence gain and 82,772 bp in sequence loss, respectively, in the KPGP9 genome relative to the hg19 reference genome. We also verified structural differences associated with TASVs by comparative analysis with TASVs in recent genomes (AK1 and TCGA genomes) and reported their details. Here, we constructed a new Korean de novo whole-genome assembly and provide the first study, to our knowledge, focused on the identification of TASVs in an individual Korean genome. Our findings again highlight the role of TEs as a major driver of structural variations in human individual genomes.

Improved High-Throughput Sequencing of the Human Oral Microbiome: From Illumina to PacBio

Background. A comprehensive understanding of the commensal microflora and its relation to health is essential for preventing and combating diseases. The aim of this study was to examine the structure of the oral microbiome by using different sequencing technologies. Material and Methods. Five preschool children with no symptoms of oral and systemic diseases were recruited. Samples of saliva were collected. A 468 bp insert size library was constructed on the MiSeq platform and then subjected to 300 bp paired-end sequencing. Libraries with longer insert sizes, including a full-length 16S rDNA gene, were sequenced on the PacBio RS II platform. Results. A total of 122.6 Mb of raw data, including 244,967 high-quality sequences, were generated by the MiSeq platform, while 134.6 Mb of raw data, including 70,030 high-quality reads, were generated by the PacBio RS II platform. Clustering of the unique sequences into OTUs at 3% dissimilarity resulted in an average of 225 OTUs on the MiSeq platform; however, the number of OTUs generated on the PacBio RS II platform was 449, far greater than the number of OTUs generated on the MiSeq platform. A total of 437 species belonging to 10 phyla and 60 genera were detected by the PacBio RS II platform, while 163 species belonging to 12 phyla and 72 genera were detected by the MiSeq platform. Conclusions. The oral microflora of healthy Chinese children were analyzed. Compared with traditional 16S rRNA sequencing technology, the PacBio system, despite providing a lower amount of clean data, surpassed the resolution of the MiSeq platform by improving the read length and annotating the nucleotide sequences at the species or strain level. This trial is registered with NCT02341352.