High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing
July 2021
Authors:
Ashley Jones ,Cynthia Torkel,David Stanley,Jamila Nasim,Justin Borevitz,Benjamin Schwessinger
Info:
The authors provide a protocol for preparing high molecular weight DNA sequencing libraries with a particular focus on difficult plant and fungal tissues, and also can be used with animals and microbes. The libraries can be used for PacBio or Oxford Nanopore sequencing, includes clean up and size selection recommendations, in a manner that is economical and scalable.
The PippinHT was used for the DNA size selection recommendations.
Author Affiliations:
Australian National University, Canberra, Australian Capital Territory, Australia
PLOS ONE
DOI: 10.1371/journal.pone.0253830
Comparison of long-read sequencing technologies in interrogating bacteria and fly genomes
June 2021
Authors:
Eric S Tvedte, Mark Gasser, Benjamin C Sparklin, Jane Michalski, Carl E Hjelmen, J Spencer Johnston, Xuechu Zhao, Robin Bromley, Luke J Tallon, Lisa Sadzewicz, David A Rasko, Julie C Dunning Hotopp
Info:
This side-by-side comparison of PacBio and Oxford Nanopore sequencing platforms for the analysis of E.coli and Drosophila assemblies. The authors provide useful guidance for which platform or approach is better suited for different areas of study, as each platform can provide certain advantages. PacBio’s Sequel II CLR, Sequel II HiFi, and RS II were evaluated as was Oxford Nanopore’s Rapid Sequencing and Ligation Sequencing. Hybrid approaches using Illumina sequencing were also evaluated.
BluePippin was used to size select libraries for both platforms.
Author Affiliations:
Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD
Department of Biology, Texas A&M University, College Station, TX
Department of Entomology, Texas A&M University, College Station, TX
G3 Genes|Genomes|Genetics
DOI:10.1093/g3journal/jkab083
Evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology
April 2021
Authors:
Multiple authors, the SEQC2 Oncopanel Sequencing Working Group
Info:
A multi-site and cross-platform evaluation of circulating tumor DNA (ctDNA) NGS assays was conducted by the SEQC2 Oncopanel Sequencing Working Group and reported on in this comprehensive study. Five assays were tested for accuracy, sensitivity and reproducibility including simulations with spike-in DNA. This study is intended to report the state-of-the-art of these assays and inform best practices.
Pippin Prep was used to size select the ctDNA reference samples using 110 bp start and 190 bp end settings for an average fragment length of ~150 bp.
Nature Biotechnology
DOI: 10.1038/s41587-021-00857-z
Nanopore adaptive sampling: a tool for enrichment of low abundance species in metagenomic samples
May 2021
Authors:
Samuel Martin, Darren Heavens, Yuxuan Lan, Samuel Horsfield, Matthew D Clark, Richard M Leggett
Info:
Oxford Nanopore adaptive sampling is a real-time sequencing method with which analysis software identifies several hundred sequence bases as they pass through a pore and decides whether to continue sequencing. If not, the pore voltage is reversed and the associated DNA molecule is ejected. The authors suggest using this method to detect rare or non-abundant species in metagenomic studies of microbial communities. To this end, they created a synthetic mock microbial consortium with seven species, presented different proportions, and tested the method. The authors demonstrate the benefit of this approach and provide a mathematical model to test the suitability for adaptive sampling for environmental metagenomic studies.
BluePippin was used to High-Pass to size select (>15kb) the DNA from the microbial samples prior to Oxford Nanopore library construction.
Author Affiliations:
Earlham Institute, Norwich, UK
Natural History Museum, London
bioRxiv preprint
DOI: 10.1101/2021.05.07.443191
Gene by environment interaction mouse model reveals a functional role for 5-hydroxymethylcytosine in neurodevelopmental disorders
May 2021
Authors:
Ligia A. Papale, Andy Madrid, Qi Zhang, Kailei Chen, Lara Sak, Sündüz Keles, and Reid S. Alisch
Info:
Researchers studying the role of 5-Hydroxymethylcytosine (5hmC) epigenetic modifications in neurological disorders report on the effect of prenatal stress on 5hMC abundance in mice. Knockouts of the Cntnap2 gene in mice have shown disruption in 5hmC, resulting in behavioral disorders. The researchers subjected mice that were heterozygous for Cntnap2, which are behaviorally normal, to prenatal stress. As adults these mice exhibited similar abnormalities. Using NGS and ChIP-seq, the authors propose a mechanism by which 5hmC alters the binding of the transcription factor CLOCK near the promoters of several genes.
PippinHT size select ChIP libraries (~400 bp) generated by the NEBNext® ChIP-Seq Library Prep Kit.
Author Affiliations:
Department of Neurological Surgery,
Department of Statistics, Biostatistics, and Medical Informatics,
Department of Neuroscience training program, University of Wisconsin, Madison, WI
Department Mathematics and Statistics, University of New Hampshire, Durham, NH
bioRxiv preprint
DOI: 10.1101/2021.05.04.441625