scholarly journals Long-Read Metagenomics to Retrieve High-Quality Metagenome-Assembled Genomes from Canine Feces

2020 ◽  
Author(s):  
Anna Cusco ◽  
Daniel Perez ◽  
Joaquim Viñes ◽  
Olga Francino
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Dna Extraction ◽  
Rrna Genes ◽  
Trna Genes ◽  
High Quality ◽  
High Molecular Weight Dna ◽  
Fecal Microbiome ◽  
Long Read ◽  
Gene Similarity

Abstract Background. Metagenomics is a powerful and rapidly developing approach that provides new biological insights into the microbes inhabiting underexplored environments, such as canine fecal microbiome. We investigate long-read metagenomics with Nanopore sequencing to profile the fecal microbiome and to retrieve high-quality metagenome-assembled genomes (HQ MAGs) from a healthy dog.Results. More than 99% of total classified reads corresponded to Bacteria. The most abundant phylum was Bacteroidetes (~80% of total reads), followed by Firmicutes, Proteobacteria, and Fusobacteria. Prevotella (>50%) and Bacteroides (>20%) are the more abundant genera, followed by Fusobacterium, Megamonas, Sutterella, and other fecal-related genera, (each representing <5% of the total bacterial composition). We retrieved eight single-contig HQ MAGs and three medium-quality MAGs, after combining several metagenome dataset assemblies. The HQ MAGs corresponded to Succinivibrio, Sutterella, Prevotellamassilia, Phascolarctobacterium, Enterococcus, Blautia, and Catenibacterium genera. Succinivibrio HQ MAG represents a novel candidate bacterial species. Sutterella HQ MAG is potentially the first reported genome assembly for Sutterella stercoricanis, as assigned by 16S rRNA gene similarity. Prevotellamassilia, Phascolarctobacterium, Catenibacterium, and Blautia sp900541345 HQ MAGs improved the contiguity of previously reported genome assemblies in their respective genera, and the number of rRNA genes and tRNA genes. Finally, Enterococcus hirae and Blautia sp003287895 HQ MAGs represented species that already have a complete reference genome. At the technical level, we demonstrated that a high-molecular weight DNA extraction improved the taxonomic classification of the raw unassembled reads, the metagenomics assembly contiguity, and the retrieval of longer and circular contigs, which are potential HQ MAGs. Conclusions. Long-read metagenomics allowed us to recover HQ MAGs from canine feces of a healthy dog. The high-molecular weight DNA extraction to improve contiguity and the correction of the insertions and deletions to reduce frameshift errors ensure the retrieval of complete single-contig HQ MAGs.

scholarly journals Optimisation of DNA extraction from the crustaceanDaphnia

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2004 ◽  
Author(s):  
Camila Gonçalves Athanasio ◽  
James K. Chipman ◽  
Mark R. Viant ◽  
Leda Mirbahai
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Dna Extraction ◽  
Organic Contaminants ◽  
High Throughput Sequencing ◽  
Model Organisms ◽  
High Yield ◽  
High Quality ◽  
High Molecular Weight Dna ◽  
Mate Pair

Daphniaare key model organisms for mechanistic studies of phenotypic plasticity, adaptation and microevolution, which have led to an increasing demand for genomics resources. A key step in any genomics analysis, such as high-throughput sequencing, is the availability of sufficient and high quality DNA. Although commercial kits exist to extract genomic DNA from several species, preparation of high quality DNA fromDaphniaspp. and other chitinous species can be challenging. Here, we optimise methods for tissue homogenisation, DNA extraction and quantification customised for different downstream analyses (e.g., LC-MS/MS, Hiseq, mate pair sequencing or Nanopore). We demonstrate that ifDaphnia magnaare homogenised as whole animals (including the carapace), absorbance-based DNA quantification methods significantly over-estimate the amount of DNA, resulting in using insufficient starting material for experiments, such as preparation of sequencing libraries. This is attributed to the high refractive index of chitin inDaphnia’scarapace at 260 nm. Therefore, unless the carapace is removed by overnight proteinase digestion, the extracted DNA should be quantified with fluorescence-based methods. However, overnight proteinase digestion will result in partial fragmentation of DNA therefore the prepared DNA is not suitable for downstream methods that require high molecular weight DNA, such as PacBio, mate pair sequencing and Nanopore. In conclusion, we found that the MasterPure DNA purification kit, coupled with grinding of frozen tissue, is the best method for extraction of high molecular weight DNA as long as the extracted DNA is quantified with fluorescence-based methods. This method generated high yield and high molecular weight DNA (3.10 ± 0.63 ng/µg dry mass, fragments >60 kb), free of organic contaminants (phenol, chloroform) and is suitable for large number of downstream analyses.

scholarly journals Scalable high-molecular weight DNA extraction for long-read sequencing v1 (protocols.io.bnjhmcj6)

protocols.io ◽  
2020 ◽  
Author(s):  
Ashley Jones ◽  
Cynthia Torkel ◽  
David Stanley ◽  
Jamila Nasim ◽  
Justin Borevitz ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Dna Extraction ◽  
High Molecular Weight Dna ◽  
Long Read

scholarly journals Extraction of high molecular weight abaca DNA suitable for next-generation sequencing

2020 ◽  
Author(s):  
Rhosener Bhea Lu Koh ◽  
Cris Francis Cortez Barbosa ◽  
Vermando Masinsin Aquino ◽  
Leny Calano Galvez
Keyword(s):  
Molecular Weight ◽  
Next Generation Sequencing ◽  
High Molecular Weight ◽  
Dna Extraction ◽  
High Throughput ◽  
Next Generation ◽  
High Quality ◽  
High Molecular Weight Dna ◽  
Ctab Method ◽  
Generation Sequencing

Abstract Background The abaca (Musa textilis Née) is a fiber crop native to the Philippines with high economic value because of its fiber - the Manila hemp, known to be the strongest of all the natural fibers. DNA extraction in abaca is difficult due to its fibrous nature, high cellulose content and polyphenol compounds. Thus an optimized DNA extraction method is required for extracting high quality abaca DNA for next-generation sequencing applications. Results In this study, we have compared five different methods for the extraction of high molecular weight DNA from abaca leaves. The methods are the traditional CTAB method (Protocol 1), the CTAB with PVP method (Protocol 2), the CTAB with 0.3% β-mercaptoethanol method (Protocol 3), SDS-method (Protocol 4) and CTAB with Triton X-100 and PVP method (Protocol 5). Out of the five methods tested, traditional CTAB-method (Protocol 1), CTAB with 0.3% β-mercaptoethanol method (Protocol 3) and SDS-method (Protocol 4) have shown to be the most consistent in giving high molecular weight DNA with good yield and purity based on A260/A280 and A260/A230 absorption values. TissueLyserII was also utilized for homogenization for the three extraction protocols for applications in high-throughput DNA extraction. DNA from two abaca varieties were extracted using the CTAB with 0.3% β-mercaptoethanol method (Protocol 3) and were sent for NGS based on Illumina HiSeq platform having both passed the quality control for library preparation. Conclusion The CTAB with 0.3% β-mercaptoethanol method (Protocol 3) was found to be the simplest and most consistent method for extracting average yield DNA with high quality for NGS applications. The SDS-method (Protocol 4) was determined to have the shortest processing time and together with TissueLyserII is the most appropriate method for high-throughput extraction of abaca samples which will be useful for genotyping-by-sequencing (GBS) studies.

scholarly journals Benchmarking ultra-high molecular weight DNA preservation methods for long-read and long-range sequencing

2021 ◽  
Author(s):  
Hollis A Dahn ◽  
Jacquelyn Mountcastle ◽  
Jennifer Balacco ◽  
Sylke Winkler ◽  
Iliana Bista ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Long Range ◽  
High Quality ◽  
High Molecular Weight Dna ◽  
Preservation Methods ◽  
Long Read ◽  
Genome Assemblies ◽  
Ultra High Molecular Weight ◽  
Chromosome Level

Studies in vertebrate genomics require sampling from a broad range of tissue types, taxa, and localities. Recent advancements in long-read and long-range genome sequencing have made it possible to produce high-quality chromosome-level genome assemblies for almost any organism. However, adequate tissue preservation for the requisite ultra-high molecular weight DNA (uHMW DNA) remains a major challenge. Here we present a comparative study of preservation methods for field and laboratory tissue sampling, across vertebrate classes and different tissue types. We find that no single method is best for all cases. Instead, the optimal storage and extraction methods vary by taxa, by tissue, and by down-stream application. Therefore, we provide sample preservation guidelines that ensure sufficient DNA integrity and amount required for use with long-read and long-range sequencing technologies across vertebrates. Our best practices generated the uHMW DNA needed for the high-quality reference genomes for Phase 1 of the Vertebrate Genomes Project (VGP), whose ultimate mission is to generate chromosome-level reference genome assemblies of all ~70,000 extant vertebrate species.

scholarly journals High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0253830
Author(s):  
Ashley Jones ◽  
Cynthia Torkel ◽  
David Stanley ◽  
Jamila Nasim ◽  
Justin Borevitz ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Dna Extraction ◽  
Magnetic Bead ◽  
Read Length ◽  
High Molecular Weight Dna ◽  
Size Selection ◽  
Oxford Nanopore ◽  
Long Read ◽  
Selection For

Rapid advancements in long-read sequencing technologies have transformed read lengths from bps to Mbps, which has enabled chromosome-scale genome assemblies. However, read lengths are now becoming limited by the extraction of pure high-molecular weight DNA suitable for long-read sequencing, which is particularly challenging in plants and fungi. To overcome this, we present a protocol collection; high-molecular weight DNA extraction, clean-up and size selection for long-read sequencing. We optimised a gentle magnetic bead based high-molecular weight DNA extraction, which is presented here in detail. The protocol circumvents spin columns and high-centrifugation, to limit DNA fragmentation. The protocol is scalable based on tissue input, which can be used on many species of plants, fungi, reptiles and bacteria. It is also cost effective compared to kit-based protocols and hence applicable at scale in low resource settings. An optional sorbitol wash is listed and is highly recommended for plant and fungal tissues. To further remove any remaining contaminants such as phenols and polysaccharides, optional DNA clean-up and size selection strategies are given. This protocol collection is suitable for all common long-read sequencing platforms, such as technologies offered by PacBio and Oxford Nanopore. Using these protocols, sequencing on the Oxford Nanopore MinION can achieve read length N50 values of 30–50 kb, with reads exceeding 200 kb and outputs ranging from 15–30 Gbp. This has been routinely achieved with various plant, fungi, animal and bacteria samples.

scholarly journals Long-read metagenomics retrieve complete single-contig bacterial genomes from canine feces

2021 ◽  
Author(s):  
Anna Cusco ◽  
Daniel Pérez ◽  
Joaquim Viñes ◽  
Norma Fàbregas ◽  
Olga Francino
Keyword(s):  
Molecular Weight ◽  
16S Rrna ◽  
High Molecular Weight ◽  
Dna Extraction ◽  
Bacterial Species ◽  
Ribosomal Genes ◽  
Bacterial Genomes ◽  
High Molecular Weight Dna ◽  
Long Reads ◽  
Long Read

Abstract BackgroundLong-read sequencing in metagenomics facilitates the assembly of complete genomes out of complex microbial communities. These genomes include essential biologic information such as the ribosomal genes or the mobile genetic elements, which are usually missed with short-reads. We applied long-read metagenomics with Nanopore sequencing to retrieve high-quality metagenome-assembled genomes (HQ MAGs) from a dog fecal sample.ResultsWe used nanopore long-read metagenomics and frameshift aware correction on a canine fecal sample and retrieved eight single-contig HQ MAGs, which were > 90% complete with < 5% contamination, and contained most ribosomal genes and tRNAs. At the technical level, we demonstrated that a high-molecular-weight DNA extraction improved the metagenomics assembly contiguity, the recovery of the rRNA operons, and the retrieval of longer and circular contigs that are potential HQ MAGs. These HQ MAGs corresponded to Succinivibrio, Sutterella, Prevotellamassilia, Phascolarctobacterium, Catenibacterium, Blautia, and Enterococcus genera. Linking our results to previous gastrointestinal microbiome reports (metagenome or 16S rRNA-based), we found that some bacterial species on the gastrointestinal tract seem to be more canid-specific –Succinivibrio, Prevotellamassilia, Phascolarctobacterium, Blautia_A sp900541345–, whereas others are more broadly distributed among animal and human microbiomes –Sutterella, Catenibacterium, Enterococcus, and Blautia sp003287895. Sutterella HQ MAG is potentially the first reported genome assembly for Sutterella stercoricanis, as assigned by 16S rRNA gene similarity. Moreover, we show that long reads are essential to gain biological insights that are otherwise missed in short-read MAGs catalogs, as shown by the mobilome functions detected in the long-read HQ MAGs.ConclusionsWe recovered eight single-contig HQ MAGs from canine feces of a healthy dog with nanopore long-reads. We also retrieved relevant biological insights from these specific bacterial species previously missed in public databases, such as complete ribosomal operons and mobilome functions. The high-molecular-weight DNA extraction improved the assembly's contiguity, whereas the high-accuracy basecalling, the raw read error correction, the assembly polishing, and the frameshift correction reduced the insertions and deletion errors. Both experimental and analytical steps ensured the retrieval of complete bacterial genomes.

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