The arguments are: This means that it has utility in generating an extremely rapid draft answer, but should not be relied upon for high accuracy without additional error correction steps such as nanopolish. Assembly of long, error-prone reads using repeat graphs. To further evaluate the accuracy of B-assembler, we used PCR amplification on 76 selected low-complexity locations and performed Sanger sequencing to get the sequences of these PCR amplifications. While Unicyclers used Racon and wtdbg2 introduced partial order alignment [35] to polish the assemblies, it seems there is still room to further improve the base accuracy. ERR550498and ERR550489; Salmonella (NCTC13349), accession no. Therefore, B-assembler outperformed all the other hybrid assemblers overall. Bandage allows you toview and manipulate de Druijn graphs output by Velvet or SPAdes lots of super cool features and useful applications, see the github sitefor examples. By performing this step, \(S1\) long reads with a high error rate will be corrected to some extent, which will improve the accuracy of genome assembly. The four assemblies were compared to evaluate their ability to reflect the genome structure. The library was amplified with six cycles of PCR using Kapa HiFi 2 mastermix (KK2601, Kapa Biosystems). Can Firtina JSK, Mohammed Alser, Damla Senol Cali, A. Ercument Cicek, Can Alkan, Onur Mutlu: Apollo. These overlaps are areas of the assembly that cannot be resolved because there are multiple identical or nearly identical sequences (kmers) in the genome, and the assembler cannot decide which sequence is attached to which other sequence. Real-time, portable genome sequencing for Ebola surveillance. Detecting recombination in whole genome comparisons. ONT library was prepared using a Rapid Sequencing Kit (SQK-RAD004) and run on a MinION Flow Cell (R9.4). Translating the Oxford Nanopore MinION sequencing technology into medical microbiology requires on-going analysis that keeps pace with technological improvements to the instrument and release of associated analysis software. When on the normal command line: tmux a -t session_name. Velvet The first and most widely used de Bruijn graph assembler built to tackle the problem of short reads. If hits are found, the sequence will be shifted based on the best hit so that the genome begins with the dnaA gene and on the forward strand. Detailed QUAST assembly metrics can be found in Additional file 1 Table S4. Interestingly, although B-assembler pipeline used Flye as the core assembly engine, B-assembler was faster and consumed less memory than Flye. Citation: Tritt A, Eisen JA, Facciotti MT, Darling AE (2012) An Integrated Pipeline for de Novo Assembly of Microbial Genomes. Compeau PE, Pevzner PA, Tesler G. How to apply de Bruijn graphs to genome assembly. This repository contains supplemental data and code for our paper: Completing bacterial genome assemblies with multiplex MinION sequencing. Nat Methods. ACT (Artemis Comparison Tool)Visualises BLAST (or similar) comparisons of genomes. Almost 50%, 80% and 90% of MinION pass reads were generated in the first 6, 9 and 12 h, respectively. A total of 20,978 ONT reads (208X) and 2.8M 150bp2 paired-end Illumina reads (7900X) were generated. 5 If it is important to know the exact termini of the genome, then the choice of sequencing is a critical factor and deciding this . The Oxford Nanopore MinION is a commercially available long read sequencer that connects to a personal computer through a USB port. Assembly using MinION data only was undertaken using PBcR (Koren et al., 2012), Canu (Berlin et al., 2015) and miniasm (Li, 2016). Unlike linear genome assemblers, Unicycler can circularize replicons without the need of postprocessing of assembly results. When a join (merging of the two ends) is made, the complete and circular assembly is completed. There are zillions of genome browsers out there, but I still love Artemis and not just because Im from the Sanger Institute. To facilitate this, future development of B-assembler will focus on speeding up the process by employing multithreading techniques. Gurevich A, Saveliev V, Vyahhi N, Tesler G. QUAST: quality assessment tool for genome assemblies. Nat Biotechnol. were recorded for all the benchmarked algorithms (Table 1). We introduced two metrics: number of supplementary alignments and number of supplementary clusters. In the hybrid-read mode, Unicycler first builds a graph with Illumina short reads by using the short-read assembler SPAdes [22], then it creates bridges with long reads in order to resolve the repeats in the genome. # Note the redirect arrow. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK, et al. Here we present TORMES, a user-friendly pipeline for WGS analysis of bacteria from any origin generated by HTS on Illumina platforms. All four had a similar number of contigs and were more contiguous than the assembly using Illumina data alone, with SPAdes producing a single chromosomal contig. Output SAM files from BWA-MEM were converted to sorted BAM files using SAMtools v0.1.19-44428cd (Li et al., 2009). Processing time and RAM used will be affected by amount of input data, complexity of data, and genome size. Even so, starting from short-read assemblies may lead to many structural errors due to the presence of repeats that are longer than the short-read lengths. Remember that to exit the tmux terminal, you will have to type -b d. To gain an intuitive and qualitative unbderstanding of assembly quality, we will simply visualise the assemblies. Fernandez L, Cima-Cabal MD, Duarte AC, Rodriguez A, Garcia P, Garcia-Suarez MDM. The most current version of Unicycler is available on GitHub. Figures were generated using multi_act_cartoon.py (Git Hub, 2016) and MUMmer (Kurtz et al., 2004) version 3.23. Although Unicycler uses Racon [15] to improve the accuracy of contigs, it cannot alleviate the structural errors caused by repeat/duplication collapses. Does RedDog report variant calls in intergenic regions? Genome Res. To detect acquired genes encoding antimicrobial resistance, the de-novo assembly was compared by BLAST to a manually curated version of the ResFinder database (compiled in 2012) (Zankari et al., 2012) as described previously (Reuter et al., 2013). In this section we will use our skill on the command-line interface to create a Article In contrast, there is a relative scarcity of tools that support genome sequencing and assembly routines. The assembly and annotation is available online (Data citation 3). We also calculated the differences (indels and mismatches) between the PCR amplicons and the contigs. 2) At the final step, it uses short reads to polish the circular contig. doi: 10.1371/journal.pone.0144305. We then evaluated the assembly of all (pass and fail) MinION reads using miniasm and Canu to determine whether adding additional (lower-quality) data would improve the assembly. B-assembler has two modes: long-read-only assembly and hybrid reads assembly. Unicycler [ 20] is one of most frequently used tools for bacterial genome assembly, and it has three modes of input: long-read-only, Illumina-only, and hybrid reads. Gillespie SH, Ling CL, Oravcova K, Pinheiro M, Wells L, Bryant JM, McHugh TD, Bebear C, Webster D, Harris SR, et al. DNA was extracted using the QiaAMP DNA Mini kit (Qiagen), and quantified using the Qubit fluorimeter (Life Technologies). The single most important thing to remember about tmux is that to do anything to control the window, you must type -b first. GALAXY is a powerful graphical open-source code-free bioinformatics platform that is freely available on multiple public and private servers. Here, we use a multidrug-resistant Enterobacter kobei isolate as a model organism to compare open source software for the assembly of genome data, and relate this to the time taken to generate actionable information. Generating an ePub file may take a long time, please be patient. To quit htop, type q. Finotello F. et al. To date, the technology has shown promise for microbiological applications, including the delineation of position and structure of bacterial antibiotic-resistance islands (Ashton et al., 2014), assembly of bacterial genomes (Loman et al., 2015; Risse et al., 2015) and tracking of viral outbreaks (Quick et al., 2016; Zika Real time Sequencing Consortium, 2016). This article has been published as part of BMC Genomics Volume 23 Supplement 4, 2022: Selected articles from the International Conference on Intelligent Biology and Medicine (ICIBM 2021): genomics. We defined a supplementary cluster as more than 10 supplementary alignments enriched in the same region. To use tmux, simply type tmux at the command prompt. You will encounter some To-do sections at times. Darling AC, Mau B, Blattner FR, Perna NT. Benchmarked on both synthetic and real sequencing data of several species of bacterium, the results show that both long-read-only and hybrid-read modes can accurately assemble circular bacterial genomes free of structural errors and have fewer small errors compared to other assemblers. PacBio or ONT reads cannot fully cover these regions, which can lead to fragmentary or incorrect assemblies. This is likely due to its homopolymer-compressed binning approach, which is less sensitive in repetitive regions, but wtdbg2 was the fastest algorithm and consumed the least memory. It has been detected in both healthy and diseased animal hosts (mouse, goat, monkey, etc.) Comparison between (a) Canu (top), manually finished (middle) and PBcR assemblies (bottom) and (b) miniasm and nanopolish (top), manually finished (middle) and SPAdes hybrid assemblies (bottom). These are complete genomes downloaded from NCBI. In addition, only the end-reads that have high mapping quality (\(\ge\) 20) are used for reassembly. Only B-assembler could achieve the exact GC value for the long-read only data. Our analyses were based on a multidrug-resistant E. kobei isolate cultured from sewage. All the tasks were submitted via Slurm job scheduling system with 4 CPUs and a total of 60GB memory. Three software tools (PBcR, Canu and miniasm) were used to assemble MinION data and a fourth (SPAdes) was used to combine MinION and Illumina data to produce a hybrid assembly. B-assembler performs several additional steps instead of directly merging two rounds of assemblies to achieve a circular genome. 2017;27(5):72236. Second, ONT or PacBio reads have much higher error rates (indels and base errors) [13] that may cause low-quality assemblies and lead to false annotations thereafter. 2012;50(9):31335. ERR710263; Legionella (NCTC11192), accession no. They indicate that there is alignment ambiguity due to structural errors based on the fact that we do not expect to see supplementary alignments or clusters in error-free assemblies. Prokka: rapid prokaryotic genome annotation. For a successful bridging, several high-quality and bona fide alignments that can cover the unsolved repeats as well as a large portion of their flanking regions are required. Achaz G, Rocha EP, Netter P, Coissac E. Origin and fate of repeats in bacteria. The Oxford Nanopore MinION sequencing technology has several advantages for pathogen sequencing in medical microbiology, but ongoing analysis needs to keep abreast of technological improvements to the instrument and release of new analysis software. We concluded from this that adding fail data did not consistently improve assembly. This ismost useful for comparisons of two or a few genomes, and makes it easy to spot and zoom in to regions of difference. Therefore, B-assembler can obtain high-quality assemblies from long noisy reads, which are critical for downstream genomic analysis of microbes. Whats best for what task? For mapping our go-to is BWA or Bowtie2 (getting from fastq -> BAM). The 48h genomic DNA sequencing script was run in MinKNOW V0.50.2.15 using the 006 workflow. The ePub format uses eBook readers, which have several "ease of reading" features There are only four basic commands to remember: -b d (Detach from the current session and return to the normal command line), -b x (eXit from the current session and quit it to return to the normal command line). By comparing the performances of several existed short-read polishing tools apollo (v2.4.0) [25], racon (v1.4.20) [15] pilon (v1.23) [14] and NextPolish (v1.3.1) [26] (see Supplementary Table 2), we selected pilon for the final polishing in hybrid mode. In order to demonstrate the performance of B-assembler in hybrid-read assembly, we isolated an M. arginini strain and deep sequenced on both Illumina MiSeq and Oxford MinION platforms. We gratefully acknowledge Catherine Ludden, Theo Gouliouris and the staff at the wastewater treatment plant for assistance in sample collection. 2017 Jun 8;13(6):e1005595. The mapping rate, single base pair mismatches, and indels were calculated to indicate the assembly accuracy. Haghshenas E, Asghari H, Stoye J, Chauve C, Hach F. HASLR: fast hybrid assembly of long reads. BMC Genomics 14, 670, 10.1186/1471-2164-14-670 (2013). Analysis of long repeats in bacterial genomes reveals alternative evolutionary mechanisms in Bacillus subtilis and other competent prokaryotes. Prokka will be used on your own made genome (assembly). The name of the program is simply flye. Last updated on May 16, 2021. Note that here I have used, # the * wildcard character to match any file that ends in ".gfa", 5.5. metAMOS is under active development and changes quite frequently Obtaining metAMOS Acquisition of high-quality bacterial genomes is fundamental, while having in mind investigation of subtitle intraspecies variation in addition to development of sensitive species-specific tools for detection and identification of the pathogens. Insights from 20 years of bacterial genome sequencing. Judge, K., Hunt, M., Reuter, S., Tracey, A., Quail, M. A., Parkhill, J. 5.1. Contact. However, many of these programs have been designed and optimised for bacterial metagenomes, which share many assembly challenges of viromes but to a lesser degree. Indels and mismatches produced by the benchmarked assemblers on the 14 NCTC PacBio samples. Second, it partitions the long reads of varied lengths into two groups: longer and shorter. DNA extraction and library preparation was performed as previously described (Quail et al., 2012). However,mostcurrent TGSassemblerswere specificallydesigned for human or other speciesthat do nothave acircular genome. Remember that the command in seqkit that gives you a summary of your .fastq file data is seqkit stats. Combining these two data types is therefore an affordable means to dramatically increase the quality of any bacterial de novo genome assembly, regardless of their genome complexity or %GC content, and compares favorably to the cost of PacBio sequencing. However, the SPAdes assembly misassembled this region into the chromosomal contig (5 Mb). document.getElementById( "ak_js_1" ).setAttribute( "value", ( new Date() ).getTime() ); Your email address will not be published. MinION and Illumina sequence data have been deposited in the European Nucleotide Archive (Data citation 1). A gap5 database was made using corrected MinION pass reads from the Canu pipeline and Illumina reads. As a comparison, we also ran wtdbg2, Flye, Canu, and Unicycler long-read-only on this dataset (Table 2). Assemblies were annotated (Seeman, 2014) and the annotation searched for the housekeeping genes rpoB and hemB (Hoffmann & Roggenkamp, 2003). The reference genome used for the simulation was the strain HAZ 145_1 [27] downloaded from the NCBI database. The command you will use will be similar to: To repeat: the -s options needs to be the same value for file 1 and file 2 to sample the reads that match with each other. Even though the size of the M. arginini genome is small (678,592bp), it contains pervasive tandem repeats, which create a challenge for effective genome assembly. Epub 2014 Dec 8. Next, some calculations. Below I am writing the command over two lines (and thus using \) so that you do not need to scroll. Siguier P, Perochon J, Lestrade L, Mahillon J, Chandler M. ISfinder: the reference centre for bacterial insertion sequences. Unlike most genome browsers, Artemis was custom-built for bacterial genomes, which lets face it are really quite different from humans and other eukaryotes. The QUAST results showed that the miniasm and nanopolish assembly had a similar number of indels per kb to Canu, although it still had more mismatches per kb (Table 1). The difference between long-read-only and hybrid modes is that since the Illumina reads have higher accuracy, B-assembler takes advantage of short reads instead of long reads for polishing and therefore can achieve more accurate assembly results. 2022 Genome Assembly of MRSA using Oxford Nanopore MinION . This suggests that this genome contains regions hard to assemble for the other assemblers. Illumina 2250bp paired-end Miseq reads that covered~250X of the genome were simulated using ART [31], with a mean insert size of 300bp and 10bp standard deviation. The polishing procedure repeats three times to achieve a whole genome sequence with the minimal errors. Loman N. J., Quick J., Simpson J. T.(2015). The read lengths range from 0.1kbp to 130kbp with a mean of 10kbp. Clinical use also requires the generation of data in a timeframe that can inform medical decisions. Note that we have updated the tutorial (as of July 2017) to version 2,available here. Therefore, it accepts PacBio, ONT, Illumina data, or a combination of them. Importantly only two libraries are needed for the hybrid assembly of a bacterial genome. Bioinformatics. The datasets together with the following accession numbers: Bacillus (NCTC3610), accession no. Wick RR, Judd LM, Gorrie CL, Holt KE. Koren S., Schatz M. C., Walenz B. P., Martin J., Howard J. T., Ganapathy G., Wang Z., Rasko D. A., McCombie W. R., et al.(2012). ngrok minecraft bedrock server; casey murphy baseball; Supplementary text, tables and figures supporting the main text. In 2013, Kat and I wrote what turned out to be a very popular Beginner's guide for comparative bacterial genome analysis. ERR832407; Salmonella (NCTC12419), accession no. First, B-assembler can reconstruct circular bacterial genomes, while other genome assemblers except Unicycler cannot achieve this goal for bacterial genome assembly by design. B-assembler is designed for ONT/Pacbio long-read only or hybrid reads (ONT/PacBio and Illumina) assembly (Fig. Most of these tools are for assembled data, hence we start with how to assemble your data this will become less of an issue as we move to long read sequencing with PacBio and MinION etc, but for the moment most of the data I work with isfromlarge scale sequencing projects with Illumina (100s-1000s) so we use mapping-based approaches for a lot of tasks so I have included a few comments about this at the end. Introduction Illumina sequencing is routinely used to study microbial genomics because of its low cost and the high accuracy of the sequence reads generated. In. Then the end-reads are assembled into a secondary assembly. Nakano K, Shiroma A, Shimoji M, Tamotsu H, Ashimine N, Ohki S, Shinzato M, Minami M, Nakanishi T, Teruya K, Satou K, Hirano T. Hum Cell. In this work, we present a new software package, B-assembler, for circular bacterial genome de novo assembly. Publication costs are funded by a startup package of University of Alabama at Birmingham to Z.C. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. Sequencing data has been deposited in the ENA under the accession numbers ERS634378: ERR1341575 (MinION pass reads) ERR1341574 (MinION fail reads) and ERR885455 (Illumina reads), 2. Forward and reverse matches are colored green and brown, respectively. Rhoads A, Au KF. Prokka will try to annotate the bacteria based on related species and starting codons can be chosen or default of the species can be used. PLoS Comput Biol. Judge, K., Hunt, M., Reuter, S., Tracey, A., Quail, M. A., Parkhill, J. The authors declare that they have no competing interests. The views expressed in this publication are those of the author(s) and not necessarily those of the Department of Health or the Wellcome Trust. 1), validating that Canu is an improvement over its predecessor PBcR. Here, we use a multidrug-resistant Enterobacter kobei isolate as a model organism to compare open source software for the assembly of genome data, and relate this to the time taken to generate actionable information. The program you will use to perform the hybrid assembly is Unicycler. How canI view myassembly graphs? Z.C. Before The discarded sequences will be substituted by the overlapping sequences of \(L1\). As with any science, there have been advances in this time. Genome Announc. In addition, to evaluate the resource usage from each component in the workflow, we stratified and benchmarked each key component of B-assembler pipeline and recorded the performance of each step. MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island. For the base accuracy, B-assembler performed best since it can eliminate all mismatches and indels, while the other hybrid assemblers all induced some levels of mismatches or indels per 100kbp. As shown in Table 2, all the assemblers produced one complete contig from M.amphoriformes ONT sequencing data. MinION-only assemblies were of sufficient quality to detect and characterise antimicrobial resistance and could be generated rapidly during an outbreak investigation. Command-line driven and runs pretty fast (<2 hours usually on our data). You can also simply type the hoel command on one line. already built in. -, Eid J. et al. As shown in Fig. ERR688913and ERR688954; Staphylococcus (NCTC10833), accession no. SPAdes gave a better accuracy for mismatches and small indels, but created a false join that incorrectly integrated a plasmid into the chromosome. Li H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Sorry, did not see this comment! Proc Natl Acad Sci U S A. Moreover, mapping long, noisy reads to short read assembled contigs is challenging because the contig lengths are relatively short, and they often contain structural errors. Three software tools (PBcR, Canu and miniasm) were used to assemble MinION data and a fourth (SPAdes) was used to combine MinION . EuGene is an open integrative gene finder for eukaryotic and prokaryotic genomes. volume23, Articlenumber:361 (2022) Before any genome assembly, it is important to determine in advance what is required from the final genome assembly. ALLPATHS-LG and SPAdes are hybrid assemblers which take short reads and long reads as inputs. Snakemake - automation and reproducibility, Bioinformatics 2015, 10.1093/bioinformatics/btv383. In this study, we propose novel algorithms for de novo assembly of bacterial transcriptomes. For example, some bacteria that colonize the gastrointestinal tract are beneficial [3], while others are pathological and induce infectious diseases and antibiotic resistance [4]. We thank Simon Harris for his advice on assembling long-read data, and the staff at Oxford Nanopore for their technical support and advice during the MinION Access Program. Incomplete bacteria genomes will make it challenging to conduct subsequent genomic analyses, such as genome structure, genome annotation, variant discovery, comparative genomics, etc. Your email address will not be published. We determined the optimal software in terms of accuracy and speed, and showed how sequence data can be used as early as 9h into the sequencing run to generate assembled whole genomes. Abstract. Identification of acquired antimicrobial resistance genes. We have compiled a software suite, SABIA (System for Automated Bacterial Integrated Annotation), which directly connects the assembly process with annotation thus streamlining the overall process. Bandage visualises the graph of an assembly - the contigs and the connections or overlaps between the contigs; see here for an explanation. The running time and memory usage of B-assembler was much lower than Unicycler. As Next-generation sequencing becomes more common, so will complete genome assemblies, enabling new research into genome structure. However, analysing plasmid content remains difficult due to incomplete assembly of plasmids. Except hybridSPAdes, B-assembler, Unicycler hybrid mode, haslr, and lathe all generated one complete contig. J Comput Biol. The advent and popularity of Third-Generation Sequencing (TGS) enables assembly of bacteria genomes at an unprecedented speed. All the tools were tested on Cheaha, the High-Performance Computer Server of the University of Alabama. S1), giving one chromosome and eleven confirmed plasmids. Genome assembly is the basis and prerequisite for understanding the genomic and functional characterization of organisms. Hunt M, Silva ND, Otto TD, Parkhill J, Keane JA, Harris SR. Circlator: automated circularization of genome assemblies using long sequencing reads. Bethesda, MD 20894, Web Policies Please do not use more than two threads! You can check how many Mbp of data you have right now by using the program that you installed previously, seqkit. 2020;9(12):916. B-assembler also demonstrated the best overall performance in resolving genome duplication sequences (dup. in Table 3). Besser J, Carleton HA, Gerner-Smidt P, Lindsey RL, Trees E. Next-generation sequencing technologies and their application to the study and control of bacterial infections. Received 2016 May 24; Accepted 2016 Aug 16. Table 1 Summary of results on 14 bacterial genome assemblies Full size table Assembly Assembly of our panel of bacterial genomes using HGAP produced a total of 71 contigs, of which 10 represented complete chromosome sequences and a further 12 represented complete plasmid sequences. Nucleic Acids Res. PLoS ONE. a file of reads (trimmed) Viewing your genome The Artemis Genome Browser. [SALZBERG2012], Assessment of de novo assemblers for draft genomes: a case study with fungal genomes. Land M, Hauser L, Jun SR, Nookaew I, Leuze MR, Ahn TH, Karpinets T, Lund O, Kora G, Wassenaar T, et al. It performs de novo genome assembly using short reads, long reads, or a hybrid method using both short and long reads. A sequencing-technology-independent, scalable, and accurate assembly polishing algorithm. DNAPlotter (alternatively circos) Software Installation Canu. Then, it collects the reads overlapping with the ends of the initial contig. You can do the same; if so, press following the \. We will use a program called Flye to build a long-read genome assembly. You will need to calculate this number. Let the secondary assembly be \(L2\). conceptualized the project. ERR550486, ERR550480 and ERR581143; Salmonella (NCTC13348), accession no. Evaluation and Validation of Assembling Corrected PacBio Long Reads for Microbial Genome Completion via Hybrid Approaches. Lee C, Grasso C, Sharlow MF. As shown in Additional file 1 Table S4, the numbers of mismatch and indel errors dropped as the pipeline ran. These results indicate that B-assembler is also capable of assembling PacBio bacterial genomes with less base errors. The NCTC also provided assembled references for these strains that were generated both automatically and manually. Thus, we used QUAST to evaluate the performance of these assemblers on this dataset. This is an open access article under the terms of the, MinION, antimicrobial resistance, assembly, software, long reads, plasmid, antimicrobial resistance, assembly, software, long reads, {"type":"entrez-nucleotide-range","attrs":{"text":"FKLS01000001-FKLS01000010","start_term":"FKLS01000001","end_term":"FKLS01000010","start_term_id":"1029966239","end_term_id":"1029965600"}}, Comparison between (a) Canu (top), manually finished (middle) and PBcR assemblies (bottom) and (b) miniasm and nanopolish (top), manually finished (middle) and SPAdes hybrid assemblies (bottom).
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