scholarly journals High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing v1 (protocols.io.bss7nehn)

protocols.io ◽  
2021 ◽  
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 ◽  
Size Selection ◽  
Long Read ◽  
Selection For

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 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 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 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.

scholarly journals Something old, something new, a DNA preparation procedure for long read genomic sequencing

Genome ◽  
2021 ◽  
Author(s):  
Hongjun Wang ◽  
Sen Xu
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Dna Extraction ◽  
Preparation Procedure ◽  
Genomic Sequencing ◽  
High Molecular Weight Dna ◽  
Short Read ◽  
Dna Preparation ◽  
Long Read ◽  
High Yields

Extracting high molecular weight DNA is critical for successful long read DNA sequencing. Here we present a DNA preparation procedure combining CTAB DNA extraction protocol and a short read eliminator kit, which consistently produces high yields of high molecular weight DNA in the freshwater microcrustacean Daphnia. This method only requires common chemicals and equipment, providing researchers a viable alternative to DNA extraction kits that often require specialized tools.

scholarly journals High molecular weight DNA extraction strategies for long-read sequencing of complex metagenomes

2021 ◽  
Author(s):  
Florian Trigodet ◽  
Karen Lolans ◽  
Emily Fogarty ◽  
Alon Shaiber ◽  
Hilary G. Morrison ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Dna Extraction ◽  
Cultured Cells ◽  
High Molecular Weight Dna ◽  
Human Tongue ◽  
Long Read ◽  
Hmw Dna ◽  
High Concentrations ◽  
Assembly Performance

AbstractBy offering extremely long contiguous characterization of individual DNA molecules, rapidly emerging long-read sequencing strategies offer comprehensive insights into the organization of genetic information in genomes and metagenomes. However, successful long-read sequencing experiments demand high concentrations of highly purified DNA of high molecular weight (HMW), which limits the utility of established DNA extraction kits designed for short-read sequencing. Challenges associated with input DNA quality intensify further when working with complex environmental samples of low microbial biomass, which requires new protocols that are tailored to study metagenomes with long-read sequencing. Here, we use human tongue scrapings to benchmark six HMW DNA extraction strategies that are based on commercially available kits, phenol-chloroform (PC) extraction, and agarose encasement followed by agarase digestion. A typical end goal of HMW DNA extractions is to obtain the longest possible reads during sequencing, which is often achieved by PC extractions as demonstrated in sequencing of cultured cells. Yet our analyses that consider overall read-size distribution, assembly performance, and the number of circularized elements found in sequencing results suggest that non-PC methods may be more appropriate for long-read sequencing of metagenomes.

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