scholarly journals Evaluation of whole-genome DNA methylation sequencing library preparation protocols

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Jacob Morrison ◽  
Julie M. Koeman ◽  
Benjamin K. Johnson ◽  
Kelly K. Foy ◽  
Ian Beddows ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bioinformatic Analysis ◽  
Limiting Factor ◽  
Whole Genome ◽  
Library Preparation ◽  
Sequencing Data ◽  
Bioinformatic Pipeline ◽  
Sequencing Cost ◽  
Sequencing Library ◽  
Sequencing Library Preparation

Abstract Background With rapidly dropping sequencing cost, the popularity of whole-genome DNA methylation sequencing has been on the rise. Multiple library preparation protocols currently exist. We have performed 22 whole-genome DNA methylation sequencing experiments on snap frozen human samples, and extensively benchmarked common library preparation protocols for whole-genome DNA methylation sequencing, including three traditional bisulfite-based protocols and a new enzyme-based protocol. In addition, different input DNA quantities were compared for two kits compatible with a reduced starting quantity. In addition, we also present bioinformatic analysis pipelines for sequencing data from each of these library types. Results An assortment of metrics were collected for each kit, including raw read statistics, library quality and uniformity metrics, cytosine retention, and CpG beta value consistency between technical replicates. Overall, the NEBNext Enzymatic Methyl-seq and Swift Accel-NGS Methyl-Seq kits performed quantitatively better than the other two protocols. In addition, the NEB and Swift kits performed well at low-input amounts, validating their utility in applications where DNA is the limiting factor. Results The NEBNext Enzymatic Methyl-seq kit appeared to be the best option for whole-genome DNA methylation sequencing of high-quality DNA, closely followed by the Swift kit, which potentially works better for degraded samples. Further, a general bioinformatic pipeline is applicable across the four protocols, with the exception of extra trimming needed for the Swift Biosciences’s Accel-NGS Methyl-Seq protocol to remove the Adaptase sequence.

Evaluation of Whole-Genome DNA Methylation Sequencing Library Preparation Protocols

2021 ◽  
Author(s):  
Jacob Morrison ◽  
Julie M. Koeman ◽  
Benjamin K. Johnson ◽  
Kelly K. Foy ◽  
Wanding Zhou ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bioinformatic Analysis ◽  
Systematic Evaluation ◽  
Whole Genome ◽  
Library Preparation ◽  
Sequencing Data ◽  
Sequencing Cost ◽  
Sequencing Library ◽  
Fresh Frozen ◽  
Sequencing Library Preparation

Abstract Background: With rapidly dropping sequencing cost, the popularity of whole-genome DNA methylation sequencing has been on the rise. Multiple library preparation protocols exist, but a systematic evaluation and benchmarking of their performance against each other is currently lacking. We have performed 22 whole-genome DNA methylation sequencing experiments on fresh frozen human samples, and extensively benchmarked common library preparation protocols for whole-genome DNA methylation sequencing, including three traditional bisulfite-based protocols and a new enzyme-based protocol. Additionally, different input DNA quantities were compared for two kits compatible with a reduced starting quantity. In addition, we also present bioinformatic analysis pipelines for sequencing data from each of these library types. Results: An assortment of metrics were collected for each kit, including raw read statistics, library quality and uniformity metrics, cytosine retention, and CpG beta value consistency between technical replicates. Overall, the NEBNext Enzymatic Methyl-seq kit performed quantitatively better than the other three protocols at two different DNA input amounts. Additionally, the results for the different input amounts were generally consistent across all metrics. Conclusions: Based on these results, we recommend use of the NEBNext Enzymatic Methyl-seq kit for whole-genome DNA methylation sequencing. Further, a general bioinformatic pipeline is applicable across the four protocols, with the exception of extra trimming needed for the Swift Bioscience's Accel-NGS Methyl-Seq protocol to remove the Adaptase sequence.

scholarly journals Methyltransferase-directed orthogonal tagging and sequencing of miRNAs and bacterial small RNAs

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Milda Mickutė ◽  
Kotryna Kvederavičiūtė ◽  
Aleksandr Osipenko ◽  
Raminta Mineikaitė ◽  
Saulius Klimašauskas ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Regulatory Networks ◽  
Library Preparation ◽  
Rna Seq ◽  
Basic Principles ◽  
Cofactor Binding ◽  
Sequencing Library ◽  
Sequencing Library Preparation ◽  
Target Rna

Abstract Background Targeted installation of designer chemical moieties on biopolymers provides an orthogonal means for their visualisation, manipulation and sequence analysis. Although high-throughput RNA sequencing is a widely used method for transcriptome analysis, certain steps, such as 3′ adapter ligation in strand-specific RNA sequencing, remain challenging due to structure- and sequence-related biases introduced by RNA ligases, leading to misrepresentation of particular RNA species. Here, we remedy this limitation by adapting two RNA 2′-O-methyltransferases from the Hen1 family for orthogonal chemo-enzymatic click tethering of a 3′ sequencing adapter that supports cDNA production by reverse transcription of the tagged RNA. Results We showed that the ssRNA-specific DmHen1 and dsRNA-specific AtHEN1 can be used to efficiently append an oligonucleotide adapter to the 3′ end of target RNA for sequencing library preparation. Using this new chemo-enzymatic approach, we identified miRNAs and prokaryotic small non-coding sRNAs in probiotic Lactobacillus casei BL23. We found that compared to a reference conventional RNA library preparation, methyltransferase-Directed Orthogonal Tagging and RNA sequencing, mDOT-seq, avoids misdetection of unspecific highly-structured RNA species, thus providing better accuracy in identifying the groups of transcripts analysed. Our results suggest that mDOT-seq has the potential to advance analysis of eukaryotic and prokaryotic ssRNAs. Conclusions Our findings provide a valuable resource for studies of the RNA-centred regulatory networks in Lactobacilli and pave the way to developing novel transcriptome and epitranscriptome profiling approaches in vitro and inside living cells. As RNA methyltransferases share the structure of the AdoMet-binding domain and several specific cofactor binding features, the basic principles of our approach could be easily translated to other AdoMet-dependent enzymes for the development of modification-specific RNA-seq techniques.

Abstract 556: Highly complex DNA sequencing library preparation for cfDNA enrichment panels using a single-stranded approach

2021 ◽  
Author(s):  
Charles J. Vaske ◽  
Chris Troll ◽  
Camille Schwartz ◽  
Colin Naughton ◽  
Abdullah Mahmood Ali ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Library Preparation ◽  
Sequencing Library ◽  
Sequencing Library Preparation

scholarly journals A Bayesian approach for analysis of whole-genome bisulphite sequencing data identifies disease-associated changes in DNA methylation

2016 ◽  
Author(s):  
Owen J.L. Rackham ◽  
Sarah R. Langley ◽  
Thomas Oates ◽  
Eleni Vradi ◽  
Nathan Harmston ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Epigenetic Modification ◽  
Promoter Hypermethylation ◽  
Whole Genome ◽  
Sequencing Data ◽  
Accurate Identification ◽  
Bisulphite Sequencing ◽  
Consistent Change ◽  
Rat Strain ◽  
Differential Transcription

ABSTRACTDNA methylation is a key epigenetic modification involved in gene regulation whose contribution to disease susceptibility remains to be fully understood. Here, we present a novel Bayesian smoothing approach (called ABBA) to detect differentially methylated regions (DMRs) from whole-genome bisulphite sequencing (WGBS). We also show how this approach can be leveraged to identify disease-associated changes in DNA methylation, suggesting mechanisms through which these alterations might affect disease. From a data modeling perspective, ABBA has the distinctive feature of automatically adapting to different correlation structures in CpG methylation levels across the genome whilst taking into account the distance between CpG sites as a covariate. Our simulation study shows that ABBA has greater power to detect DMRs than existing methods, providing an accurate identification of DMRs in the large majority of simulated cases. To empirically demonstrate the method’s efficacy in generating biological hypotheses, we performed WGBS of primary macrophages derived from an experimental rat system of glomerulonephritis and used ABBA to identify >1,000 disease-associated DMRs. Investigation of these DMRs revealed differential DNA methylation localized to a 600bp region in the promoter of the Ifitm3 gene. This was confirmed by ChIP-seq and RNA-seq analyses, showing differential transcription factor binding at the Ifitm3 promoter by JunD (an established determinant of glomerulonephritis) and a consistent change in Ifitm3 expression. Our ABBA analysis allowed us to propose a new role for Ifitm3 in the pathogenesis of glomerulonephritis via a mechanism involving promoter hypermethylation that is associated with Ifitm3 repression in the rat strain susceptible to glomerulonephritis.

scholarly journals Extraction and high-throughput sequencing of oak heartwood DNA: Assessing the feasibility of genome-wide DNA methylation profiling

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0254971
Author(s):  
Federico Rossi ◽  
Alessandro Crnjar ◽  
Federico Comitani ◽  
Rodrigo Feliciano ◽  
Leonie Jahn ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Tree Ring ◽  
High Throughput ◽  
Environmental Conditions ◽  
High Throughput Sequencing ◽  
Wood Formation ◽  
Whole Genome ◽  
Library Preparation ◽  
Bisulfite Treatment ◽  
Standing Trees

Tree ring features are affected by environmental factors and therefore are the basis for dendrochronological studies to reconstruct past environmental conditions. Oak wood often provides the data for these studies because of the durability of oak heartwood and hence the availability of samples spanning long time periods of the distant past. Wood formation is regulated in part by epigenetic mechanisms such as DNA methylation. Studies of the methylation state of DNA preserved in oak heartwood thus could identify epigenetic tree ring features informing on past environmental conditions. In this study, we aimed to establish protocols for the extraction of DNA, the high-throughput sequencing of whole-genome DNA libraries (WGS) and the profiling of DNA methylation by whole-genome bisulfite sequencing (WGBS) for oak (Quercus robur) heartwood drill cores taken from the trunks of living standing trees spanning the AD 1776-2014 time period. Heartwood contains little DNA, and large amounts of phenolic compounds known to hinder the preparation of high-throughput sequencing libraries. Whole-genome and DNA methylome library preparation and sequencing consistently failed for oak heartwood samples more than 100 and 50 years of age, respectively. DNA fragmentation increased with sample age and was exacerbated by the additional bisulfite treatment step during methylome library preparation. Relative coverage of the non-repetitive portion of the oak genome was sparse. These results suggest that quantitative methylome studies of oak hardwood will likely be limited to relatively recent samples and will require a high sequencing depth to achieve sufficient genome coverage.

scholarly journals Rapid construction of a whole-genome transposon insertion collection for Shewanella oneidensis by Knockout Sudoku

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Michael Baym ◽  
Lev Shaket ◽  
Isao A. Anzai ◽  
Oluwakemi Adesina ◽  
Buz Barstow
Keyword(s):  
Single Gene ◽  
Shewanella Oneidensis ◽  
Whole Genome ◽  
Sequencing Data ◽  
Kinetic Measurements ◽  
Data Set ◽  
Quinone Reduction ◽  
Transposon Insertion ◽  
Sequencing Library ◽  
Technical Effort

Abstract Whole-genome knockout collections are invaluable for connecting gene sequence to function, yet traditionally, their construction has required an extraordinary technical effort. Here we report a method for the construction and purification of a curated whole-genome collection of single-gene transposon disruption mutants termed Knockout Sudoku. Using simple combinatorial pooling, a highly oversampled collection of mutants is condensed into a next-generation sequencing library in a single day, a 30- to 100-fold improvement over prior methods. The identities of the mutants in the collection are then solved by a probabilistic algorithm that uses internal self-consistency within the sequencing data set, followed by rapid algorithmically guided condensation to a minimal representative set of mutants, validation, and curation. Starting from a progenitor collection of 39,918 mutants, we compile a quality-controlled knockout collection of the electroactive microbe Shewanella oneidensis MR-1 containing representatives for 3,667 genes that is functionally validated by high-throughput kinetic measurements of quinone reduction.

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